Cytotoxicity mediation of cells evidencing surface expression of CD63

ABSTRACT

This invention relates to the diagnosis and treatment of cancerous diseases, particularly to the mediation of cytotoxicity of tumor cells; and most particularly to the use of cancerous disease modifying antibodies (CDMAB), optionally in combination with one or more chemotherapeutic agents, as a means for initiating the cytotoxic response. The invention further relates to binding assays which utilize the CDMABs of the instant invention.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/810,751,filed Mar. 26, 2004, which is a continuation-in-part of application Ser.No. 10/603,006, filed Jun. 23, 2003, which is a continuation-in-part ofapplication Ser. No. 10/348,231, filed Jan. 21, 2003, now U.S. Pat. No.7,009,040, issued Mar. 7, 2006, the contents of each of which are hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to the diagnosis and treatment of cancerousdiseases, particularly to the mediation of cytotoxicity of tumor cells;and most particularly to the use of cancerous disease modifyingantibodies (CDMAB), optionally in combination with one or morechemotherapeutic agents, as a means for initiating the cytotoxicresponse. The invention further relates to binding assays, which utilizethe CDMAB of the instant invention.

BACKGROUND OF THE INVENTION

CD63 is a Type III membrane protein of the tetraspanin family whose 20current members are characterized by the presence of four transmembranesegments. Several groups independently identified CD63, using antibodiesraised to whole cell preparations of activated platelets, granulocytes,and melanoma cells. Cloning of the respective cDNAs of their cognateglycoprotein antigens led to the recognition that the different antigenswere one and the same molecule. The Sixth International Workshop onLeukocyte Typing (1996) subsequently categorized these antibodies asCD63 antibodies. Prior to the 1996 Workshop, CD63 was known by multiplenames (melanoma 1 antigen, ocular melanoma-associated antigen, melanomaassociated antigen ME491, lysosome-associated membrane glycoprotein 3,granulophysin, melanoma-associated antigen MLA1), which were sometimesrelated to the antibodies that led to its partial characterization andidentification. Thus, CD63 was also designated as antigen ME491 (MAbME491), neuroglandular antigen (MAbs LS59, LS62, LS76, LS113, LS140 andLS152), Pltgp40 (MAbs H5C6, H₄F₈ and H5D2), human bone marrow stromalcell antigen (MAb 12F12), osteoprogenitor-specific marker (MAb HOP-26),and integrin-associated protein (MAb 6H1). Other antibodies that werefound to cross react with human CD63 were 8-1H, 8-2A (cross-reactivitywith ME491), NKI/C-3 and NKI/black-13 (Vannegoor and Rumke, 1986;Demetrick et al., 1992; Wang et al., 1992).

CD63 was initially cloned from a melanoma cDNA library using MAb ME491,one of a number of antibodies raised against a preparation of humanmelanoma cells. It was shown that the reactivity of MAb ME491 appearedto be inversely correlated with melanoma progression in a study of humanmelanoma biopsies. The reactivity of the ME491 antibody was low innormal melanocytes, higher in the early stages of melanoma progression(dysplastic nevi and radial growth phase (RGP) tumors) and decreased oreven absent in more advanced melanoma tumors such as those in thevertical growth phase (VGP) and in metastatic tumors.

CD63 was also found and partially characterized in human platelets usingMAb 2.28 (raised against activated platelets) that detected anactivation-dependent platelet membrane 53 kDa glycoprotein. Thismolecule was also associated with the membrane of internal granules inunstimulated platelets. In the same study MAb 2.28 also labelledinternal granules in megakaryocytes and endothelial cells, where itco-localized with antibodies to the enzyme cathepsin D, a known markerof lysosomal compartments. Follow up studies with antibody clusteringand expression cloning, led to the identification of the antigenrecognized by this antibody as CD63, and further confirmed its presencein lysosomal compartments, where it co-localized with thecompartment-specific markers LAMP-1 and LAMP-2. Cloning of this moleculeidentified it as CD63 and allowed its inclusion in the tetraspaninfamily.

Expression of CD63 was detected in many different tissues and celltypes. At the cellular level it was found to be associated with theplasma membrane and also with intracellular late endosomal vesicularstructures. Cell activation led, in certain cases, to increased surfaceexpression by mobilization of intracellular stores of CD63. CD63 wasalso found to co-localize, and physically associate, with MHC class IIin B-lymphocytes, particularly in endosomes, in exosomes involved inexporting MHC class II complexes to the surface, and in secretedvesicles. CD63 was found to interact with other members of thetetraspanin family, such as CD9, CD81, CD11 (integrin chain α_(M,L,X)),CD18 (integrin chain β₂), CD49c (VLA-3 or integrin chain α₃), CD49d(integrin chain α₄), CD49f (VLA-6 or integrin chain α₆) and CD29(integrin chain β₁), in a variety of cell types including B- andT-lymphocytes, neutrophils, breast cancer and melanoma cells.

The role of CD63 in cancer has been unclear. Although CD63 was initiallydiscovered by several independent groups to be involved in diverseevents such as platelet and granulocyte activation, MHC classII-dependent antigen presentation, integrin-dependent cell adhesion andmotility, and tumor progression in certain types of cancers, itsfunction has yet to be fully elucidated. Even though current evidencesupports its role in a variety of cellular physiological events, it isnot clear if these functions are independent of each other or if thereis an underlying common cellular mechanism in which CD63 is involved.

Several groups have investigated the association between CD63 and theprogression of certain types of tumors, particularly melanomas. A numberof other anti-CD63 monoclonal antibodies, in addition to Mab ME491, weredeveloped for immunohistochemical (IHC) staining of cancer samplesobtained from patients with tumors at various stages of progression. Itwas observed that decreased staining, interpreted by the authors as mostlikely reflecting decreased expression of CD63, correlated with advancedprogression and with metastatic characteristics of the tumors. A morerecent study, also described a significant correlation between theapparent decreased expression levels (after quantitation of mRNA) ofseveral members of the tetraspanin protein family, including CD63, andthe in vitro invasiveness of several mammary carcinoma-derived celllines. Another study identified CD63, by differential display, incultured breast cancer cells subjected to estrogen deprivation. Thisindicated that CD63 expression can be steroid-hormone regulated and thataltered CD63 abundance and/or function might also be associated withbreast tumor progression.

By contrast, work with anti-CD63 monoclonal antibody MAb FC-5.01revealed that its reactive epitope was variably expressed in differentnormal tissues. Although this antibody was found to recognize CD63, itdid not distinguish between early and more advanced stage melanomas,including metastatic melanomas (unlike MAb ME491), which suggested thatthe CD63 antigen was present in these more advanced tumors, but thatsome of its epitopes may have been masked in the cells from tumors atdifferent stages. This might have been due to altered post-translationalmodifications of the core CD63 polypeptide, or to the interaction ofCD63 with other molecules, which might have affected the availability ofspecific epitopes for antibody recognition and binding. These resultssupported the observation, described by Si and Hersey (1993), thatstaining with the anti-CD63 MAb NKI-C3, did not distinguish betweentissue sections from melanomas at different stages of progression, suchas primary, radial growth phase, vertical growth phase, and metastaticmelanomas. Although in other studies (Adachi et al., 1998; Huang et al.,1998) analysis of mRNA from breast, and from non-small-cell lungcancers, by quantitative PCR, revealed that for two tetraspanin familymembers (CD9 and CD82) there was a significant correlation between theirexpression levels and tumor progression and patient prognosis, no suchcorrelation was found for CD63, in that its expression was similar inall the samples. As a result of these, apparently conflicting, results,there is lack of strong and consistent data that would definitivelydemonstrate the association of CD63 with cancer.

To date very few in vivo studies have attempted to establish a linkbetween CD63 and an eventual tumor suppressor function of this molecule.In one of these studies, human CD63-overexpressing H-ras-transformedNIH-3T3 cells, injected both subcutaneously and intraperitoneally intoathymic mice, revealed a decreased malignant/tumorigenic phenotype, asindicated by decreased tumor size and metastatic potential as well as byincreased survival time, when compared to the behavior of the parentalnon-CD63-overexpressing cells. This suggested that the presence of humanCD63 in the transformed cells might suppress their malignant behavior.More recently, work with a transgenic mouse model expressing human CD63,and developed to induce tolerance to CD63, indicated that tumor growthof an injected human CD63-MHC class I (H-2K^(b)) co-transfected murinemelanoma cell line could be inhibited, and survival increased, uponimmunization with human CD63 fused to vaccinia virus. It was suggestedby the authors that the therapeutic effect was T-lymphocyte dependent,and that endogenous anti-CD63 antibodies did not appear to be involvedin this protective effect, since tumor growth inhibition only occurredwhen animals were injected with the CD63-MHC class I co-transfectedcells and not with the CD63-only transfected cell line. Thisinterpretation was supported by the fact that in wild type animals,pre-immunized with purified human CD63 and shown to have developedanti-human CD63 antibodies, there was no protective effect against tumorcell growth. Work described by Radford et al. (1995) using the KM3 cellline, initially thought to be of human origin but later characterized asbeing of rat lineage, transfected with human CD63, suggested thatexpression of this protein decreased the growth and metastasticpotential of these cells, relative to that observed using the parentalnon-transfected KM3 cells, when injected intradermally into athymicmice, although there was no significant difference between the in vitrogrowth rates of the various transfected and non-transfected cell lines.These observations distinguished the potential effect of CD63 from thatof other tumor suppressor genes known to affect both the in vivo and thein vitro growth rates of tumor cells. Furthermore, addition of theanti-CD63 monoclonal antibody ME491, which was found to have afunctional effect on the same cells by decreasing their random motilityin an in vitro assay (Radford et al., 1997), did not impact their invitro growth rates.

This study also described the observation that CD63 may promotemigration in response to extracellular matrix (ECM)-derivedchemoattractants, such as laminin, fibronectin, collagen andvitronectin, and that this effect may be mediated by the functionalinvolvement of β₁-type integrins, although antibodies to the integrinswere unable to block these effects. However, there appeared to be anantagonistic effect between the role of vitronectin-mediated signaling(a known ligand for the integrin α_(v)β₅) and that of the signalingmediated by other ECM components such as fibronectin, laminin andcollagen on CD63 transfected cells. This suggested that under specificconditions, in the presence of ECM components, expression of CD63 maylead to decreased migration, and that this may be dependent on a finebalance between adhesion and motility. In another study, an anti-CD63monoclonal antibody (MAb 710F) enhanced the adhesion and spreading ofPMA-treated HL-60 cells, while another anti-CD63 monoclonal antibody(MAb 2.28), promoted a similar effect, but only on a much smallerfraction of the cell population, and only when added in much largeramounts. These results showed that although many antibodies to CD63 havebeen developed, their functional effects can be quite different.

Tetraspanins may also be involved in cell proliferation. Oren et al.(1990) described anti-proliferative effects of the murine MAb 5A6, thatrecognizes CD81 (TAPA-1), on lymphoma cell lines. In another study,ligation of CD37 in human T-lymphocytes with antibodies blockedCD37-induced proliferation. More recently, a study with an animal modeldeficient in the expression of CD37 (CD37 knockout) revealed that Tlymphocytes from this animal were hyperproliferative compared to thosefrom wild type animals in response to concanavalin A activation andCD3/T cell receptor engagement. It was therefore proposed that afunctional role in cell growth and proliferation might be a commonfeature of the tetraspanin family. Recent studies with hepatoblastomaand hepatocellular carcinoma cells revealed that engagement of thesecells with anti-CD81 monoclonal antibodies led to activation of theErk/MAP kinase pathway. This signaling pathway has been shown to beinvolved with cell growth and proliferation events. In parallel work,transfected cell lines overexpressing human CD81 displayed increasedproliferation relative to the mock-transfected control cells. Therefore,available evidence has pointed to a role of the tetraspanins in general,and of CD63 in particular, in events associated with cell growthproliferation and with cell adhesion/motility. These two types ofcellular events are currently the target of intense research as bothplay a central role in tumor progression and metastasis.

Until now, no anti-CD63 antibodies, or other reagents that specificallytargeted CD63-expressing cells, were reported and shown to have asimultaneous impact on the in vitro and on the in vivo growthcharacteristics of tumor cells, and also on the survival time of animalmodels of tumor cell growth.

Amino acid sequence determination and analysis did not reveal homologybetween tetraspanins and other protein families, or with any previouslycharacterized functional modules, nor has it suggested any previouslyknown enzymatic activity. As a result it has been very difficult toinvestigate the role of this family of proteins in the modulation ofsignal transduction pathways. However, the evidence generated usingtetraspanin-specific reagents that led to changes in cellularphysiology, and which were intimately dependent on the modulation ofsignal transduction pathways, suggests that tetraspanins have signaltransduction properties. CD63 was shown to associate, both physicallyand functionally, with a number of molecules that are themselves eitherenzymes involved in the generation of secondary messenger signals, orare associated physically and/or functionally with such enzymes.

Experiments designed to dissect the mechanism controlling theinteraction of human neutrophils with endothelial cells, which is one ofthe initial steps of the inflammatory response, revealed thatpre-treatment of neutrophils with several anti-CD63 monoclonalantibodies (AHN-16, AHN-16.1, AHN-16.2, AHN-16.3 and AHN-16-5) promotedtheir adhesion to cultured endothelial cell layers. Furthermore thiseffect was strongly dependent on the presence of calcium ion (Ca²⁺), awell-known modulator of many intracellular signaling pathways and whichwas restricted to a specific period of time during which the cells wereexposed to the stimulating antibodies. After longer exposure to theantibody, adhesion of the neutrophils to the endothelial cells becameinsensitive to the later addition of Ca²⁺, therefore implicating adynamic and temporally regulated (transitory) event. In addition, CD63was found to physically interact with the CD11/CD18 protein complex, andreagents that specifically targeted this complex mediated a modulatorysignal. In this study CD63 was also found to be physically associatedwith, or to be part of, a complex that included the enzyme tyrosinekinases Lck and Hck. These enzymes are members of a class of proteinsthat play a central role in mediating intracellular regulatory signalsupon activation of specific surface receptors and are part of cascadesof signaling pathways that result in cell-specific physiologicalchanges. Another study suggested that co-ligation of tetraspanins(including CD63) with monoclonal antibodies could enhance thephosphorylation or activity of the enzyme focal adhesion kinase (FAK)that was induced by adhesion of MDA-MB-231 breast cancer cells tocollagen substrate. This pointed to a direct involvement of CD63 (and ofother tetraspanin family members) in the modulation of integrin-mediatedtyrosine kinase signaling pathways. Other signaling pathways that mayfunctionally intersect with the presence and ligation of surface CD63 bythe anti-CD63 monoclonal antibody MAb 710F appear to be those dependenton modulation of phosphorylation by the enzyme protein kinase C (PKC),another well known modulator of intracellular signaling pathways. Inthis context, enhancement of adhesion and of morphological changes inthe myeloid cell line HL-60 by MAb 710F was dependent on pre-treatmentof the cells with phorbol myristate acetate (PMA) although the temporalinvolvement of PKC was not conclusively demonstrated. However, laterwork by an independent group demonstrated that PMA-induced HL-60differentiation was PKC-activity dependent since the molecule Ro31-8220,a specific inhibitor of this enzyme, blocked the effect of PMA.

Further evidence supporting the association of CD63, and othertetraspanin family members, with signal transduction pathways, arosefrom work that described a physical association, either direct or aspart of a supramolecular complex, between CD63 (and also CD53) moleculeswith tyrosine phosphatase activity. In this study, immunoprecipitatecomplexes isolated with anti-CD63 antibodies were shown to be associatedwith tyrosine phosphatase activity, although unlike for CD53, which wasshown to associate with the tyrosine phosphatase CD45, it was notpossible to identify the CD63-associated phosphatase. More recentlyseveral members of the tetraspanin family were also found to beassociated with a type II phosphatidylinositol 4-kinase (type II PI 4-K)(Berditchevski et al., 1997). This interaction appeared to be veryspecific since it was only identified for CD9, CD63, CD81, CD151 andA15/TALLA, and it was not observed to occur with CD37, CD52, CD82, orNAG-2. In addition, the association between tetraspanin family membersand PI-4K was mutually exclusive since each PI-4 kinase-containingcomplex was limited to a single tetraspanin family member. CD63-PI-4kinase complexes, in particular, were found, almost entirely, inintracellular compartments in lipid raft-like domains, unlike thoseformed with the other tetraspanin members. This observation suggestedthat this CD63 fraction, found to interact with the PI-4 kinase, mighthave been involved in specific intracellular events (Claas, C, et al.,2001) related to, or dependent from, phosphoinositide biosynthesispathways, which are well known for their involvement in the regulationof membrane trafficking (endocytosis and exocytosis) and of cytoskeletonreorganization, in addition to their function as secondary messengermolecules (Martin, T., 1998).

The direct and important involvement of all the enzymes, that CD63 wasfound until now to be directly associated with, in the regulation ofsignaling pathways provided further evidence in support of theassociation of CD63 with the modulation of signal transduction pathways,either as a regulator or as an effector molecule downstream from theactivity of these enzymes.

Elucidation of the mechanisms that lead to tumor progression is a verydifficult and complex endeavor frequently marked by apparentlycontradictory observations and, as a result, it rare that thoseobservations successfully translate into effective therapies. In view ofwhat is currently known about the association of CD63 with tumorprogression and metastasis and with signal transduction mechanisms, itis possible that its function may be altered, in tumor cells.

Development of antigen-specific reagents with cytotoxic effects on tumorcells, that bind cells expressing the recognized antigen(s) and which bythemselves, or associated with other molecules, have cellular and invivo physiological activity such that these reagents inhibit tumor cellgrowth, progression and metastasis, without significant deleteriouseffects on normal cell populations, would be extremely beneficial as apotential therapeutic and or diagnostic tool.

Prior Patents

U.S. Pat. No. 5,296,348 teaches methods for selecting monoclonalantibodies specific for cancer cell surface antigens that areinternalizing, and for identifying monoclonal antibodies havinganti-transcriptional and/or anti-replicational effects on cellmetabolism. By way of example the ME491 antibody was shown tointernalize in W9, WM35, WM983 melanoma cells, and SW948 colorectalcarcinoma cells. In addition ME491 antibody was shown to decreasetranscription and cell proliferation in SW948 cells. The patentapplication US20030211498A1 (and its related applications: WO0175177A3,WO0175177A2, AU0153140A5) allege a method of inhibiting the growth ormetastasis of an ovarian tumor with an antibody that binds an ovariantumor marker polypeptide encoded by an ovarian tumor marker geneselected from among a group that includes CD63 antigen. Serial analysisof gene expression using ovarian cancer was carried out to identifyovarian tumor marker genes which lead to the identification of CD63 as acandidate. The patent application WO02055551A1 (and its relatedapplication CN1364803A) alleges a new polypeptide-human CD63 antigen56.87. The patent application CN1326962A alleges a new polypeptide-humanCD63 antigen 14.63. The patent application CN1326951A alleges a newpolypeptide-human CD63 antigen 15.07. The patent application CN1351054Aalleges a new polypeptide-human CD63 antigen 11.11. These patents andpatent applications identify CD63 antigens and antibodies but fail todisclose the isolated monoclonal antibody of the instant invention, orthe utility of the isolated monoclonal antibody of the instantinvention.

SUMMARY OF THE INVENTION

The instant inventors have previously been awarded U.S. Pat. No.6,180,357, entitled “Individualized Patient Specific Anti-CancerAntibodies” directed to a process for selecting individually customizedanti-cancer antibodies, which are useful in treating a cancerousdisease. For the purpose of this document, the terms “antibody” and“monoclonal antibody” (mAb) may be used interchangeably and refer tointact immunoglobulins produced by hybridomas (e.g. murine or human),immunoconjugates and, as appropriate, immunoglobulin fragments andrecombinant proteins derived from said immunoglobulins, such as chimericand humanized immunoglobulins, F(ab′) and F(ab′)₂ fragments,single-chain antibodies, recombinant immunoglobulin variable regions(Fv)s, fusion proteins etc. It is well recognized in the art that someamino acid sequence can be varied in a polypeptide without significanteffect on the structure or function of the protein. In the molecularrearrangement of antibodies, modifications in the nucleic or amino acidsequence of the backbone region can generally be tolerated. Theseinclude, but are not limited to, substitutions (preferred areconservative substitutions), deletions or additions. Furthermore, it iswithin the purview of this invention to conjugate standardchemotherapeutic modalities, e.g. radionuclides, with the CDMAB of theinstant invention, thereby focusing the use of said chemotherapeutics.The CDMAB can also be conjugated to toxins, cytotoxic moieties, enzymese.g. biotin conjugated enzymes, or hematogenous cells, thereby formingan antibody conjugate.

This application utilizes the method for producing patient specificanti-cancer antibodies as taught in the '357 patent for isolatinghybridoma cell lines which encode for cancerous disease modifyingmonoclonal antibodies. These antibodies can be made specifically for onetumor and thus make possible the customization of cancer therapy. Withinthe context of this application, anti-cancer antibodies having eithercell-killing (cytotoxic) or cell-growth inhibiting (cytostatic)properties will hereafter be referred to as cytotoxic. These antibodiescan be used in aid of staging and diagnosis of a cancer, and can be usedto treat tumor metastases.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existingcancerous disease modifying antibodies. The patient will beconventionally staged but the available antibodies can be of use infurther staging the patient. The patient can be treated immediately withthe existing antibodies and/or a panel of antibodies specific to thetumor can be produced either using the methods outlined herein orthrough the use of phage display libraries in conjunction with thescreening methods herein disclosed. All the antibodies generated will beadded to the library of anti-cancer antibodies since there is apossibility that other tumors can bear some of the same epitopes as theone that is being treated. The antibodies produced according to thismethod may be useful to treat cancerous disease in any number ofpatients who have cancers that bind to these antibodies.

Using substantially the process of U.S. Pat. No. 6,180,357, and asdisclosed in Ser. No. 10/348,231, the mouse monoclonal antibody7BD-33-11A was obtained following immunization of mice with cells from apatient's breast tumor biopsy. The 7BD-33-11A antigen was expressed onthe cell surface of a wide range of human cell lines from differenttissue origins. The breast cancer cell line MCF-7 and prostate cancercell line PC-3 were susceptible to the cytotoxic effects of 7BD-33-11Ain vitro.

The result of 7BD-33-11A cytotoxicity against breast and prostate cancercells in culture was further extended by its anti-tumor activity towardsthese cancer indications in vivo (as disclosed in Ser. No. 10/348,231and Ser. No. 10/603,006). Pre-clinical xenograft tumor models areconsidered valid predictors of therapeutic efficacy.

As outlined and described in Ser. No. 10/348,284 and Ser. No.10/603,006, 7BD-33-11A prevented tumor growth and tumor burden in apreventative in vivo model of human breast cancer. Monitoring continuedpast 300 days post-treatment. 7BD-33-11A never developed tumors and 87.5percent of the 7BD-33-11A treatment group was still alive at over 9months post-implantation. Conversely, the isotype control group had 100percent mortality by day 72 (23 days post-treatment). Therefore7BD-33-11A enhanced survival and prevented tumor growth (thus delayingdisease progression) in a breast cancer model.

Also as outlined and described in Ser. No. 10/348,284 and Ser. No.10/603,006, 7BD-33-11A significantly suppressed tumor growth anddecreased tumor burden in an established in vivo model of human breastcancer. By day 80 (23 days post-treatment), 7BD-33-11A treated mice had83 percent lower mean tumor volumes in comparison to the isotype controlgroup (p=0.001). Using survival as a measure of antibody efficacy, itwas estimated that the risk of dying in the 7BD-33-11A treatment groupwas about 16 percent of the isotype control group (p=0.0006) at around60 days post-treatment. 100 percent of the isotype control group died by50 days post-treatment. In comparison, 60 percent of the 7BD-33-11Atreatment groups were still alive at 130 days post-treatment. This datademonstrated that 7BD-33-11A treatment conferred a survival benefit andreduced tumor burden compared to the control treated group. 7BD-33-11Atreatment appeared safe, as it did not induce any signs of toxicity,including reduced body weight and clinical distress. Thus, 7BD-33-11Atreatment was efficacious as it both delayed tumor growth and enhancedsurvival compared to the control-treated groups in a well-establishedmodel of human breast cancer.

The effect of 7BD-33-11A compared to a chemotherapeutic drug (Cisplatin)treatment alone or in combination was determined in two differentestablished breast cancer xenograft models. In the MDA-MB-231 (MB-231)model, at day 69 (5 days after treatment), 7BD-33-11A treatment resultedin a 76 percent reduction in tumor growth relative to the buffer controltreated animals (p<0.001). Cisplatin treatment alone or in combinationwith 7BD-33-11A resulted in a 79 and 86 percent reduction in tumor size,respectively, relative to the control (p<0.001). In the MDA-MB-468(MB-468) model, at day 55 (5 days after treatment) Cisplatin treatmentalone and in combination with 7BD-33-11A showed the greatest reductionin tumor growth, 95 percent (p=0.024) and 97 percent (p=0.17)respectively. Also at day 55, 7BD-33-11A treatment alone showed areduction in tumor growth by 37 percent (p=0.3958), in comparison to thebuffer control. In both the MB-231 and MB-468 model, treatment with7BD-33-11A led to greater animal well-being in comparison to treatmentwith Cisplatin as measured by body weight. These results indicate that7BD-33-11A treatment has greater efficacy in comparison to Cisplatintreatment alone in the MB-231 model and was better tolerated with feweradverse effects, such as weight loss, than Cisplatin in both breastcancer models.

To determine the efficacious effects of 7BD-33-11A treatment at variousdoses, a dose response experiment was performed in a preventative breastcancer xenograft model. At day 55 (5 days after treatment), the 0.2mg/kg treatment group had prevented tumor growth by 85 percent relativeto the isotype control treated group. Also at day 55, both the 2 and 20mg/kg treatment groups had yet to develop tumors. Similar results wereobtained past day 125 (75 days after treatment), where the 20 mg/kgtreatment group had still not developed tumors and the 2 mg/kg treatmentgroup had some initial tumor growth. 7BD-33-11A treatment alsodemonstrated a survival benefit. All of the mice in the isotype controlgroup had died by day 104 (54 days after treatment) while the 0.2 mg/kg7BD-33-11A treatment group survived until day 197 (147 days aftertreatment). Even greater survival benefits were observed with the 2.0and 20 mg/kg 7BD-33-11A treatment groups; only 50 percent of the 2.0mg/kg treatment group had died by day 290 (240 days after treatment)while none of the 20 mg/kg treatment group had died by also day 290.Therefore, 7BD-33-11A treatment showed significant tumor growthreduction and increased survival with all three doses with the greatestdegree of efficacy being exhibited by the highest dose.

In addition to the beneficial effects in the established in vivo tumormodel of breast cancer, 7BD-33-11A treatment also had anti-tumoractivity against PC-3 cells in a preventative in vivo prostate cancermodel (outlined in Ser. No. 10/603,006). 7BD-33-11A treatment wassignificantly (p=0.001) more effective in suppressing tumor growthshortly after the treatment period than an isotype control antibody. Atthe end of the treatment phase, mice given 7BD-33-11A had tumors thatgrew to only 31 percent of the isotype control group. For PC-3 SCIDxenograft models, body weight can be used as a surrogate indicator ofdisease progression. On day 52, 7BD-33-11A treatment significantly(p=0.002) prevented the loss of body weight by 54 percent in comparisonto isotype control. Mice were monitored for survival post-treatment. At11 days post-treatment, isotype and buffer control mice had reached 100percent mortality. Conversely, 7BD-33-11A reached 100 percent mortalityat day 38 post-treatment, 3 times longer than the control groups. Thus,7BD-33-11A treatment was efficacious as it both delayed tumor growth,prevented body weight loss and extended survival compared to the isotypecontrol treated group in a well-established model of human prostatecancer.

In addition to the preventative in vivo tumor model of prostate cancer,7BD-33-11A demonstrated anti-tumor activity against PC-3 cells in anestablished in vivo tumor model (outlined in Ser. No. 10/603,006).Treatment with 7BD-33-11A was again compared to isotype control. It wasshown that the 7BD-33-11A treatment group had significantly (p<0.024)smaller mean tumor volumes compared with the isotype control treatedgroup immediately following treatment. 7BD-33-11A treatment mediatedtumor suppression by 36 percent compared to the isotype control group.The anti-tumor activities of 7BD-33-11A, in several different cancermodels, make it an attractive anti-cancer therapeutic agent.

In order to validate the 7BD-33-11A epitope as a drug target, theexpression of 7BD-33-11A antigen in normal human tissues was previouslydetermined (Ser. No. 10/603,006). This work was extended by comparisonwith commercially available anti-CD63 antibodies (RFAC4 and H5C6).Results from tissue staining indicated that 7BD-33-11A again showedrestricted binding to various cell types but had binding to infiltratingmacrophages, lymphocytes, and fibroblasts. The RFAC4 and H5C6 antibodiesshowed a similar staining pattern in comparison to each other. However,the staining pattern of both RFAC4 and H5C6 was quite different thanthat observed with 7BD-33-11A. Specifically, both RFAC4 and H5C6antibodies bound to a broader range of normal tissues, usually hadhigher staining intensity in tissues where 7BD-33-11A was also positiveand bound not only to infiltrating macrophages, lymphocytes andfibroblasts but also to the epithelium in a majority of the tissues.

Localization of the 7BD-33-11A antigen and determining its prevalencewithin the population, such as among breast cancer patients, isimportant in assessing the therapeutic use of 7BD-33-11A and designingeffective clinical trials. To address 7BD-33-11A antigen expression inbreast tumors from cancer patients, tumor tissue samples from 50individual breast cancer patients were previously screened forexpression of the 7BD-33-11A antigen (Ser. No. 10/603,006). Current workcompared the staining of 7BD-33-11A to RFAC4 and H5C6 and to ananti-Her2 antibody (c-erbB-2). The results of the current study weresimilar to previous results and showed that 36 percent of tumor tissuesamples stained positive for the 7BD-33-11A antigen while 94 and 85percent of breast tumor tissues were positive for the H5C6 and RFAC4epitope respectively. Expression of 7BD-33-11A within patient samplesappeared specific for cancer cells as staining was restricted tomalignant cells. In addition, 7BD-33-11A stained 0 of 10 samples ofnormal tissue from breast cancer patients while both H5C6 and RFAC4stained 7 of 8 samples of normal breast tissue. Breast tumor expressionof the 7BD-33-11A antigen appeared to be localized to the cell membraneand cytoplasm of malignant cells, making CD63 an attractive target fortherapy. 7BD-33-11A expression was further evaluated based on breasttumor expression of the receptors for the hormones estrogen andprogesterone, which play an important role in the development,treatment, and prognosis of breast tumors. There was a slightcorrelation between estrogen or progesterone receptor expression andexpression of 7BD-33-11A; tissues with receptor expression had slightlyhigher 7BD-33-11A expression. When tumors were analyzed based on theirstage, or degree to which the cancer advanced, results suggested a trendtowards greater positive expression with higher tumor stage for7BD-33-11A. Similar results were obtained with RFAC4. H5C6 also showed avery slight correlation with estrogen or progesterone receptorexpression but there was no apparent correlation with tumor stage.However, for all three antibodies, the results were limited by the smallsample size. In comparison to c-erbB-2, 7BD-33-11A showed a completelydifferent staining profile where half of the breast tumor tissue samplesthat were positive for the 7BD-33-11A antigen were negative for Her2expression indicating a yet unmet targeted therapeutic need for breastcancer patients. There were also differences in the intensity ofstaining between the breast tumor tissue sections that were positive forboth 7BD-33-11A and Her2. The c-erbB-2 antibody also positively stainedone of the normal breast tissue sections.

Localization of the 7BD-33-11A antigen and its prevalence withinprostate cancer patients is important in assessing the benefits of7BD-33-11A immunotherapy to patients with prostate cancer and designingeffective clinical trials. To address 7BD-33-11A antigen expression inprostate tumors from cancer patients, tumor tissue samples from 51individual prostate cancer patients were screened for expression of the7BD-33-11A antigen. The results of the study showed that 88 percent oftissue samples stained positive for the 7BD-33-11A antigen. Although7BD-33-11A stained the normal tissue sections with high intensity aswell, there was a higher degree of membranous staining in the tumortissue samples in comparison to the normal samples. There was oneembryonal rhabdomyosarcroma tissue sample that did not stain for the7BD-33-11A antigen. There also appeared to be no direct correlationbetween tumor stage and presence of the 7BD-33-11A antigen. However, theresults were limited by the small sample size.

To further extend the potential therapeutic benefit of 7BD-33-11A, thefrequency and localization of the antigen within various human cancertissues was also previously determined (Ser. No. 10/603,006). Severalcancer types, in addition to breast and prostate cancer, expressed the7BD-33-11A antigen. The positive human cancer types included skin (1/2),lung (3/4), liver (2/3), stomach (4/5), thyroid (2/2), uterus (4/4) andkidney (3/3). Some cancers did not express the antigen; these includedovary (0/3), testis (0/1), brain (0/2) and lymph node (0/2). As withhuman breast and prostate cancer tissue, localization of 7BD-33-11Aoccurred both on the membrane and within the cytoplasm of these tumorcells. So, in addition to the 7BD-33-11A antibody binding to cancer celllines in vitro, there is evidence that the antigen is expressed inhumans, and on multiple types of cancers.

As outlined herein, additional biochemical data also indicate that theantigen recognized by 7BD-33-11A is CD63. This is supported by studiesshowing that two monoclonal antibodies (RFAC4 and H5C6), reactiveagainst CD63, identify proteins that bound to 7BD-33-11A byimmunoprecipitation. In addition, further bacterial expression studieselucidated that 7BD-33-11A bound to extracellular loop 2 of CD63. The7BD-33-11A epitope was also distinguished by being conformationdependent. These IHC and biochemical results demonstrate that 7BD-33-11Abinds to the CD63 antigen. Thus, the preponderance of evidence showsthat 7BD-33-11A mediates anti-cancer effects through ligation of aunique conformational epitope present on CD63. For the purpose of thisinvention, said epitope is defined as a “CD63 antigenic moiety”characterized by its ability to bind with a monoclonal antibody encodedby the hybridoma cell line 7BD-33-11A, antigenic binding fragmentsthereof or antibody conjugates thereof.

In toto, this data demonstrates that the 7BD-33-11A antigen is a cancerassociated antigen and is expressed in humans, and is a pathologicallyrelevant cancer target. Further, this data also demonstrates the bindingof the 7BD-33-11A antibody to human cancer tissues, and can be usedappropriately for assays that can be diagnostic, predictive of therapy,or prognostic. In addition, the cell membrane localization of thisantigen is indicative of the cancer status of the cell due to the lackof expression of the antigen in most non-malignant cells, and thisobservation permits the use of this antigen, its gene or derivatives,its protein or its variants to be used for assays that can bediagnostic, predictive of therapy, or prognostic.

The present invention describes the development and use of 7BD-33-11A,developed by the process described in patent U.S. Pat. No. 6,180,357 andidentified by, its effect, in a cytotoxic assay, in non-established andestablished tumor growth in animal models and in prolonging survivaltime in those suffering from cancerous disease. This inventionrepresents an advance in the field of cancer treatment in that itdescribes, for the first time, a reagent that binds specifically to anepitope present on the target molecule, CD63, and that also has in vitrocytotoxic properties against malignant tumor cells but not normal cells,and which also directly mediates inhibition of tumor growth andenhancement of survival in in vivo models of human cancer. This is anadvance in relation to any other previously described anti-CD63antibody, since none have been shown to have similar properties. It alsoprovides an advance in the field since it clearly demonstrates, and forthe first time, the direct involvement of CD63 in events associated withgrowth and development of certain types of tumors. It also represents anadvance in cancer therapy since it has the potential, to display similaranti-cancer properties in human patients. A further advance is thatinclusion of this antibody in a library of anti-cancer antibodies willenhance the possibility of targeting tumors expressing different antigenmarkers by determination of the appropriate combination of differentanti-cancer antibodies, to find the most effective in targeting andinhibiting growth and development of the tumors.

In all, this invention teaches the use of the 7BD-33-11A antigen as atarget for a therapeutic agent, that when administered can reduce thetumor burden of a cancer expressing the antigen in a mammal, and canalso lead to a prolonged survival of the treated mammal. This inventionalso teaches the use of a CDMAB (7BD-33-11A), and its derivatives, andantigen binding fragments thereof, to target its antigen to reduce thetumor burden of a cancer expressing the antigen in a mammal, and toprolong the survival of a mammal bearing tumors that express thisantigen. Furthermore, this invention also teaches the use of detectingthe 7BD-33-11A antigen in cancerous cells that can be useful for thediagnosis, prediction of therapy, and prognosis of mammals bearingtumors that express this antigen.

If a patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient, or a compatible donor, and re-infused for treatment ofmetastases. There have been few effective treatments for metastaticcancer and metastases usually portend a poor outcome resulting in death.However, metastatic cancers are usually well vascularized and thedelivery of anti-cancer antibodies by red blood cells can have theeffect of concentrating the antibodies at the site of the tumor. Evenprior to metastases, most cancer cells are dependent on the host's bloodsupply for their survival and anti-cancer antibodies conjugated to redblood cells can be effective against in situ tumors as well.Alternatively, the antibodies may be conjugated to other hematogenouscells, e.g. lymphocytes, macrophages, monocytes, natural killer cells,etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC). For example murine IgM andIgG2a antibodies can activate human complement by binding the C-1component of the complement system thereby activating the classicalpathway of complement activation which can lead to tumor lysis. Forhuman antibodies, the most effective complement-activating antibodiesare generally IgM and IgG1. Murine antibodies of the IgG2a and IgG3isotype are effective at recruiting cytotoxic cells that have Fcreceptors which will lead to cell killing by monocytes, macrophages,granulocytes and certain lymphocytes. Human antibodies of both the IgG1and IgG3 isotype mediate ADCC.

Another possible mechanism of antibody-mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are two additional mechanisms of antibody-mediated cancer cellkilling, which are more widely accepted. The first is the use ofantibodies as a vaccine to induce the body to produce an immune responseagainst the putative antigen that resides on the cancer cell. The secondis the use of antibodies to target growth receptors and interfere withtheir function or to down regulate that receptor so that its function iseffectively lost.

The clinical utility of a cancer drug is based on the benefit of thedrug under an acceptable risk profile to the patient. In cancer therapysurvival has generally been the most sought after benefit, however thereare a number of other well-recognized benefits in addition to prolonginglife. These other benefits, where treatment does not adversely affectsurvival, include symptom palliation, protection against adverse events,prolongation in time to recurrence or disease-free survival, andprolongation in time to progression. These criteria are generallyaccepted and regulatory bodies such as the U.S. Food and DrugAdministration (F.D.A.) approve drugs that produce these benefits(Hirschfeld et al. Critical Reviews in Oncology/Hematolgy 42:137-1432002). In addition to these criteria it is well recognized that thereare other endpoints that may presage these types of benefits. In part,the accelerated approval process granted by the U.S. F.D.A. acknowledgesthat there are surrogates that will likely predict patient benefit. Asof year-end (2003), there have been sixteen drugs approved under thisprocess, and of these, four have gone on to full approval, i.e.,follow-up studies have demonstrated direct patient benefit as predictedby surrogate endpoints. One important endpoint for determining drugeffects in solid tumors is the assessment of tumor burden by measuringresponse to treatment (Therasse et al. Journal of the National CancerInstitute 92(3):205-216 2000). The clinical criteria (RECIST criteria)for such evaluation have been promulgated by Response EvaluationCriteria in Solid Tumors Working Group, a group of international expertsin cancer. Drugs with a demonstrated effect on tumor burden, as shown byobjective responses according to RECIST criteria, in comparison to theappropriate control group tend to, ultimately, produce direct patientbenefit. In the pre-clinical setting tumor burden is generally morestraightforward to assess and document. In that pre-clinical studies canbe translated to the clinical setting, drugs that produce prolongedsurvival in pre-clinical models have the greatest anticipated clinicalutility. Analogous to producing positive responses to clinicaltreatment, drugs that reduce tumor burden in the pre-clinical settingmay also have significant direct impact on the disease. Althoughprolongation of survival is the most sought after clinical outcome fromcancer drug treatment, there are other benefits that have clinicalutility and it is clear that tumor burden reduction, which may correlateto a delay in disease progression, extended survival or both, can alsolead to direct benefits and have clinical impact (Eckhardt et al.Developmental Therapeutics: Successes and Failures of Clinical TrialDesigns of Targeted Compounds; ASCO Educational Book, 39^(th) AnnualMeeting, 2003, pages 209-219).

Accordingly, it is an objective of the invention to utilize a method forproducing cancerous disease modifying antibodies from cells derived froma particular individual which are cytotoxic with respect to cancer cellswhile simultaneously being relatively non-toxic to non-cancerous cells,in order to isolate hybridoma cell lines and the corresponding isolatedmonoclonal antibodies and antigen binding fragments thereof for whichsaid hybridoma cell lines are encoded.

It is an additional objective of the invention to teach CDMAB andantigen binding fragments thereof.

It is a further objective of the instant invention to produce CDMABwhose cytotoxicity is mediated through ADCC.

It is yet an additional objective of the instant invention to produceCDMAB whose cytotoxicity is mediated through CDC.

It is still a further objective of the instant invention to produceCDMAB whose cytotoxicity is a function of their ability to catalyzehydrolysis of cellular chemical bonds.

A still further objective of the instant invention is to produce CDMABwhich are useful in a binding assay for diagnosis, prognosis, andmonitoring of cancer.

Other objects and advantages of this invention will become apparent fromthe following description wherein, by way of illustration and example,certain embodiments of this invention are set forth.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Western blot of MDA-MB-231 whole cell lysates (Lane 1) ormembranes (Lanes 2 and 3) probed with 7BD-33-11A (Panel A) or isotypecontrol (Panel B). Molecular weight markers are indicated on the left.

FIG. 2. Western blot of MDA-MB-231 membranes probed with 7BD-33-11A.Lane 1: Membrane run under reducing conditions. Lane 2: Membranes rununder non-reducing conditions. Molecular weight markers are indicated onthe left.

FIG. 3. Effect of deglycosylation on the binding of 7BD-33-11A toMDA-MB-231 membranes. MDA-MB-231 membranes were subjected to treatmentwith glycopeptidast F (PNGase F; Lane 1), O-glycanase (Lane 2),sialidase (Lane 3), the combination of PNGase F, O-glycanase andsialidase (Lane 4), the combination of PNGase F, O-glycanase, sialidase,galactosidase and glucosaminidase (Lane 5) or buffer control (Lane 6).Molecular weight markers are indicated on the left.

FIG. 4. SDS-PAGE (Panel A) and Western blot (Panel B) of MDA-MB-231membrane proteins immunoprecipitated with 7BD-33-11A. Lane A isotypecontrol immunoprecipitated proteins, Lane B: 7BD-33-11Aimmunoprecipitated proteins and Lane TM: Total MDA-MB-231 membraneproteins. Rectangular box outlines the same band from Lane B in theSDS-PAGE and Lane TM in the Western blot. Molecular weight markers areindicated on the left.

FIG. 5. Profound search summary table.

FIG. 6. MASCOT search summary table. The peptide disclosed in the lowerright corner is designated SEQ ID NO: 5.

FIG. 7 a: Western blots of proteins probed with 7BD-33-11A (Panel A),anti-CD63 (clone RFAC4, Panel B), IgG2a isotype control (Panel C) andIgG1 isotype control (Panel D). Lane A: Total MDA-MB-231 membraneproteins; Lane B: 7BD-33-11A immunoprecipitated proteins; Lane C:anti-CD63 (RFAC4) immunoprecipitated proteins, Lane D: IgG2a isotypecontrol immunoprecipitated proteins and Lane E: IgG1 isotype controlimmunoprecipitated proteins. Molecular weight markers are indicated onthe left.

FIG. 7 b: Western blots of proteins probed with 7BD-33-11A (Panel A),anti-CD63 (clone H5C6, Panel B), IgG2a isotype control (Panel C) andIgG1 isotype control (Panel D). Lane A: Total MDA-MB-231 membraneproteins; Lane B: 7BD-33-11A immunoprecipitated proteins; Lane C:anti-CD63 (H5C6) immunoprecipitated proteins, Lane D: IgG2a isotypecontrol immunoprecipitated proteins and Lane E: IgG1 isotype controlimmunoprecipitated proteins. Molecular weight markers are indicated onthe left.

FIG. 8. Western blots of proteins probed with 7BD-33-11A (Panel A),anti-CD63 (clone RFAC4, Panel B), anti-CD63 (clone H5C6, Panel C), IgG2aisotype control (Panel D) and IgG1 isotype control (Panel E). Lanes 1-5contain 7BD-33-11A immunoprecipitated proteins and Lanes 6-10 containIgG2a isotype control immunoprecipitated proteins. Lanes 1 and 6: noNaCl, Lanes 2 and 7: 150 mM NaCl, Lanes 3 and 8: 500 mM NaCl, Lanes 4and 9: 2000 mM NaCl and Lanes 5 and 10: RIPA buffer.

FIG. 9. Western blots of proteins probed with 7BD-33-11A (Panel A),anti-CD63 (clone RFAC4, Panel B), anti-CD63 (clone H5C6, Panel C), IgG2aisotype control (Panel D) and Coomassie Colloidal Blue protein stain(Panel E). Lane 1: non-induced vector alone, Lane 2: non-inducedGST-EC1, Lane 3: non-induced GST-EC2, Lane 4: induced vector alone, Lane5: induced GST-EC1 and Lane 6: induced GST-EC2. Molecular weight markersare indicated on the left.

FIG. 10. Representative FACS histograms of 7BD-33-11A, isotype controlsand anti-EGFR directed against several cancer cell lines and non-cancercells.

FIG. 11. Representative micrographs showing the binding pattern obtainedwith 7BD-33-11A (A), isotype negative control (B), anti-CD63 (RFAC4)antibody or the anti-CD63 (H5C6) antibody (D) on tissues sections ofcolon from a normal human tissue array. 7BD-33-11A, RFAC4 and H5C6displayed positive staining for macrophages and lymphocytes at thelamina propria. RFAC4 and H5C6 also displayed strong staining for themucosal epithelieum. Magnification is 200×.

FIG. 12. Representative micrographs showing the binding pattern obtainedwith 7BD-33-11A (A), isotype negative control (B), anti-CD63 (RFAC4)antibody or the anti-CD63 (H5C6) antibody (D) on tissues sections ofinfiltrative ductal carcinoma from a human breast cancer tissue array.7BD-33-11A displayed weaker positive staining for the tumor cells incomparison to either RFAC4 or H5C6 antibody. Magnification is 200×.

FIG. 13. Representative micrographs showing the binding pattern obtainedwith 7BD-33-11A (A) or the anti-Her2 (c-erbB-2) antibody (B) on tissuessections of infiltrative ductal carcinoma from a human breast cancertissue array. 7BD-33-11A displayed strong positive staining for thetumor cells in comparison to the anti-Her2 antibody, which displayednegative staining. Magnification is 200×.

FIG. 14. Representative micrographs showing the binding pattern obtainedwith 7BD-33-11A on tissues sections of prostate adenocarinoma (A) ornormal prostate (B) from a human prostate cancer tissue array.7BD-33-11A displayed strong positive membranous staining for the tumorcells in the adenocarcinoma tissue section. 7BD-33-11A showed bothmembranous and cytoplasmic staining of the glandular epithelium in thenormal prostate tissue section. Magnification is 200×.

FIG. 15. Effect of 7BD-33-11A or isotype control on tumor growth in adose response preventative MDA-MB-231 breast cancer model. The dashedline indicates the period during which the antibody was administered.Data points represent the mean+/−SEM.

FIG. 16. Survival of tumor-bearing mice after treatment with 7BD-33-11Aor isotype control antibody in a dose response preventative MDA-MB-231xenograft study.

FIG. 17. Effect of 7BD-33-11A, Cisplatin, 7BD-33-11A+Cisplatin or buffercontrol on tumor growth in an established MDA-MB-231 breast cancermodel. The dashed line indicates the period during which the antibodywas administered. Data points represent the mean+/−SEM.

FIG. 18. Effect of 7BD-33-11A, Cisplatin, 7BD-33-11A+Cisplatin or buffercontrol on body weight in an established MDA-MB-231 breast cancer model.

FIG. 19. Effect of 7BD-33-11A, Cisplatin, 7BD-33-11A+Cisplatin or buffercontrol on tumor growth in an established MDA-MB-468 breast cancermodel. The dashed line indicates the period during which theantibody/Cisplatin was administered. Data points represent themean+/−SEM.

FIG. 20. Effect of 7BD-33-11A, Cisplatin, 7BD-33-11A+Cisplatin or buffercontrol on body weight in an established MDA-MB-468 breast cancer model.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Identification ofBinding Proteins by Western Immunoblotting

To identify the antigen(s) recognized by the antibody 7BD-33-11A, cellmembrane preparations were subjected to sodium dodecylsulphatepolyacrylamide gel electrophoresis (SDS-PAGE), and transferred tomembranes. The latter were probed with the antibody 7BD-33-11A tovisualize the proteins detected by this antibody.

1 Whole Cell Lysate and Total Membrane Fraction Preparation 1.1. WholeCell Lysate Preparation

Previous work by FACS demonstrated binding of antibody 7BD-33-11A to thebreast cancer cell line MDA-MB-231 (MB-231). As a result total cellmembrane preparations and whole cell lysates obtained from this cellline were used for the antigen identification and characterization.Total cell lysate from MB-231 cells was prepared as follows: MB-231 cellpellet (1.5 g) was resuspended in 2 mL lysis buffer containing 20 mMTris, pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100, 0.02% (w/v) sodiumazide, 2 mM sodium orthovanadate, 50 mM sodium fluoride, and a proteaseinhibitor cocktail (Roche Diagnostics; Manheim, Germany). The pellet washomogenized with a glass homogenizer and was incubated with stirring,for 1 hr at 4° C. Samples were then subjected to centrifugation (20,000g) for 15 min at 4° C., to remove detergent insoluble material.Supernatants were collected, divided in aliquots, and frozen at −80° C.The protein concentration in the cell lysate was determined by the BCA(bicinchoninic acid) assay (Pierce; Rockford, Ill.).

1.2. Total Cell Membrane Fraction Preparation

Total cell membranes were prepared from confluent cultures of MB-231breast cancer cells. Media was removed from cell stacks and the cellswere washed with phosphate buffered saline (PBS). Cells were dissociatedwith dissociation buffer (Gibco-BRL; Grand Island, N.Y.) for 20 min at37° C. on a platform shaker. Cells were collected and centrifuged at 900g for 10 min at 4° C. After centrifugation, cell pellets were washed byresuspending in PBS and centrifuging again at 900 g for 10 min at 4° C.Pellets were then stored at −80° C. until required. To preparemembranes, cell pellets were thawed and resuspended in homogenizationbuffer containing 1 tablet per 50 mL of complete protease inhibitorcocktail (Roche; Laval QC) at a ratio of 3 mL buffer per gram of cells.The cell suspension was subjected to homogenization using a polytronhomogenizer on ice in order to lyse the cells. The cell homogenate wascentrifuged at 15,000 g for 10 min at 4° C. to remove the nuclearparticulate. Supernatant was harvested, divided into tubes and thencentrifuged at 75,600 g for 90 min at 4° C. Supernatant was carefullyremoved and each membrane pellet was resuspended in approximately 5 mLof homogenization buffer. The membrane pellets from all tubes werecombined, divided one more time, and centrifuged at 75,600 g for 90 minat 4° C. Supernatant was carefully removed and the pellets were weighed.Solubilization buffer containing 1% Triton X-100 was added to thepellets at a ratio of 3 mL buffer per gram of membrane pellet. Membraneswere solubilized by shaking on a platform shaker at 300 rpm, for 1 hr onice. The membrane suspension was centrifuged at 75,600 g to pelletinsoluble material. The supernatant, containing the solubilized membraneproteins, was carefully removed from the tubes, assayed for proteinconcentration, and stored at −80° C.

2. 1-Dimensional SDS-PAGE and Western Immunoblotting

Proteins from the total membrane fraction and whole cell lysate ofMB-231 cells were separated by 1-dimensional SDS-PAGE (1D SDS-PAGE), ona 5 and 10 percent stacking and separating gel, respectively. Proteinswere transferred overnight, at 4° C., by electroblotting onto PVDFmembranes (Millipore; Billerica, Mass.). Complete transfer wasdetermined by assessing the transfer of prestained molecular weightmarkers onto the membrane. After transfer, the membranes were blockedwith 5 percent (w/v) skim milk in TBST, for 1 hr at room temperature(RT), and two replicate blots were then probed as follows: one blot wasprobed with the antibody 7BD-33-11A (5 μg/ml, in 5 percent skim milk inTBST) and the replicate blot was probed with an IgG_(2a) isotype control(5 μg/ml, in 5 percent skim milk in TBST). Blots were washed 3 times for10 min in TBST and then incubated with horseradish HRP-conjugated goatanti-mouse IgG (Fc) (Bio-Rad Laboratories; Hercules, Calif.), for 1 hrat RT. After washing 3 times for 10 min each with TBST, the blots weredeveloped with the TMB peroxidase substrate kit (Vector Laboratories;Burlingame, Calif.) following the manufacturers' instructions. The blotswere rinsed with water and images were acquired with a gel documentationsystem (FIGS. 1 and 2) (Bio-Rad; Hercules, Calif.). Blots were imagedunder the same conditions of camera focus, aperture and imageacquisition time. In FIG. 1, 7BD-33-11A clearly bound to proteins in the20-80 kDa range, and its reactivity was detected in the lanes containingwhole cell lysate and total membrane fraction. The isotype control didnot bind to any proteins in the MB-231 lysate or membrane fractions,indicating that the binding for 7BD-33-11A was specific. FIG. 2demonstrated the effect of sample reduction on 7BD-33-11A binding, on aWestern blot. Reactivity of this antibody was only detected when thesamples were prepared under non-reducing conditions (Lane 2). Reducingagents such as DTT or β-mercaptoethanol completely eliminated binding(Lane 1), indicating that recognition and binding of 7BD-33-11A to itsepitope on the native protein depended on the presence of disulfidebonds.

To determine if the disperse nature of the antigen, as detected byWestern immunoblotting, was due to heterogeneous glycosylation, totalmembrane fractions were subjected to treatment with several glycosidases(glycopeptidase F, o-glycanase, sialidase, galactosidase andglucosaminidase) which removed specific carbohydrate groups. Aftertreatment the samples were subjected to 1D SDS-PAGE and Westernblotting. It was expected that if some of the enzymes removed a portionof carbohydrate that accounted for a significant amount of the mass ofthe antigen(s) recognized by the antibody 7BD-33-11A, that it would bepossible to detect that difference by SDS-PAGE. FIG. 3 shows thatglycosidase treatment of total membrane fractions from MB-231 cellsresulted in a significant decrease in the mass of the recognizedantigen(s). This indicated that the antigen recognized by the 7BD-33-11Aantibody was comprised of at least one glycoprotein. The fact that asignificant shift in the mobility of the antigen(s) only occurred whenseveral enzymes were used together indicated that at least some of thecarbohydrate moiety consisted of a complex N-linked carbohydrate.Although treatment of the membrane with glycosidases resulted in amolecular weight shift, it did not reduce the intensity of binding. Thissuggested that the antibody bound primarily to the polypeptide portionof the glycoprotein.

Example 2 Identification of Antigens Bound by 7BD-33-11A

1. Immunoprecipitation of Antigens from MB-231 Total Membrane Fraction

Total membrane extracts (5 mg total protein) were diluted to a 1 mg/mlfinal protein concentration with the appropriate volume of 1× lysisbuffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.02% NaN₃, 2mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitorcocktail (Roche Diagnostics, Manheim, Germany)), and with theappropriate volume of 2×RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl,1.0% sodium cholate, 0.2% SDS, 1% Triton X-100 and 0.02% NaN₃), in orderto obtain a final 1×RIPA buffer concentration. The extracts werepre-cleared for 2 hr with protein G-Sepharose beads (AmershamBiosciences, Uppsala, Sweden) at 4° C. Total membrane extracts wereremoved and stock BSA (10 mg/ml) was added to a 0.5 mg/ml final BSAconcentration. While extracts were being pre-cleared,antibody-conjugated protein G-Sepharose beads (60 μg of antibodychemically cross-linked to 30 μl of protein G Sepharose) were blockedwith 1 mL of 0.5 mg/ml BSA, by incubation at 4° C., also for 2 hr. Afterblocking, the antibody-conjugated beads were washed twice for 5 min with1×RIPA buffer. The antibody-conjugated protein G-Sepharose beads werethen added to the BSA-containing total membrane extracts, and incubatedfor 3 hr, at 4° C., on an end-over-end rotator. After centrifugation at20,000 g, for 10 seconds, at 4° C., the unbound fraction was removed anddiscarded, and the beads were washed 3 times for 5 min, with 1 mL ofRIPA buffer in each wash step. The beads were then rinsed once with 1.5ml of PBS. The immunoprecipitation (IP) described above, with7BD-33-11A-conjugated protein G Sepharose was carried out in parallelwith a similar IP in which the protein G-Sepharose beads were chemicallycross-linked with an IgG2a isotype control (BD Biosciences, San Diego,Calif.). This step was carried out to enable assessment of non-specificbinding of proteins to the immunocomplexes. After completely drainingthe PBS, the beads were boiled in 40 μl of non-reducing sample bufferand the samples were analyzed by 1D SDS-PAGE followed by Westernimmunoblotting of a portion of the gel, and staining with CoomassieColloidal Blue of the remaining portion of the gel. Of the 40 μl, afraction (8 μl) was loaded onto the SDS-PAGE for Western blotting andthe remaining fraction (32 μl) was loaded onto a separate lane of thesame gel for protein staining with Coomassie Colloidal Blue. The portionof the gel designated for protein staining was incubated overnight withthe Coomassie Colloidal Blue stain. The portion of the gel designatedfor Western blotting was transferred onto a PVDF membrane for 2 hr at320 mA, rinsed with deionized water, blocked for 1 hr at RT with 5percent milk in TBST and then incubated overnight at 4° C. with7BD-33-11A in 5 percent milk in TBST. Blots were washed 3 times for 10min in TBST and incubated with an HRP-conjugated Fc-specific goatanti-mouse IgG (1:5000) in 5 percent milk in TBST, for 1 hr at roomtemperature. Blots were then washed 3 times for 10 min and weredeveloped according to the standard procedure of TMB substrate for HRP.As displayed in FIG. 4, the Western immunoblot and the CoomassieColloidal Blue stained gel were lined up, using the molecular weightmarkers as reference. The main band that stained with CoomassieColloidal Blue lined up with the main band that reacted with 7BD-33-11Aon the Western blot. This section is highlighted (rectangle inset) onFIG. 4.

2. Peptide Mapping, and Antigen Identification by Mass Spectrometry

From the experiment above, the band on the Coomassie Colloidal Bluestained gel that lined up with the most intense reactivity on theWestern blot was then cut out and subjected to in-gel tryptic digestionusing a commercially available kit (Pierce, Rockford, Ill.). Aliquots ofthe digest were subjected to mass spectrometry analysis on a SELDI-TOFCiphergen PBSIIc reader (Ciphergen Biosystems Inc., Freemont, Calif.).Briefly, an aliquot of the digest was manually spotted onto an H4 chip(Ciphergen Biosystems Inc., Freemont, Calif.). After drying, an aliquotof CHCA matrix (α-cyano 4-hydroxy cinnaminic acid; Ciphergen BiosystemsInc., Freemont, Calif.) was added onto the same spot on the chip andallowed to dry. The sample was then analyzed on the PBSIIc reader.Similar sized bands from parallel regions on isotype control lanes andblank gel region were processed side-by-side with the gel plug from the7BD-33-11A IP, so as to enable determination of unique peptide fragmentsgenerated by the digestion of the antigen immunoprecipitated by7BD-33-11A. The masses of the unique peptide fragments were searchedusing PROFOUND, a publicly accessible online tool for searching proteinsequence databases using information from mass spectra. The uniquepeptides in the sample from the 7BD-33-11A IP digest were then subjectedto MS/MS analysis on a QSTAR (Applied Biosystems, Foster City, Calif.)equipped with an interface that enabled analysis of the same samplespots that were previously analyzed on the PBSIIc reader. The MS/MS datawas then analyzed with MASCOT, a publicly accessible online tool forsearching protein databases using information from MS/MS spectra. FIG. 5is a summary of the table that resulted from the ProFound search. Theonly protein that was suggested as a putative candidate, with asignificant degree of confidence was CD63. FIG. 6 is a summary tablethat resulted from the MASCOT search. The only protein that wasidentified with a high degree of probability was CD63, supporting theprevious tentative identification by peptide map fingerprinting.

3. 7BD-33-11A Antigen ID Confirmation

Confirmation of the ID of the putative antigen for 7BD-33-11A wascarried out through determination of whether known anti-human CD63monoclonal antibodies (e.g. RFAC4 and H5C6) would react with theprotein(s) immunoprecipitated by 7BD-33-11A, and vice versa. Furtherconfirmation was also carried out by Western immunoblotting of totallysates from induced and non-induced bacteria transformed withglutathione S-transferase (GST)-fusion constructs of the extracellulardomains of human CD63. Immunoprecipitates from MB-231 total membrane,and prepared with the monoclonal antibodies 7BD-33-11A, RFAC4 (CymbusBiotechnology LTD, Hants, UK), H5C6 (BD Biosciences, San Diego, Calif.),and with the IgG_(2a) and IgG₁ (BD Biosciences, San Diego, Calif.)isotype controls, were analyzed by 1D SDS-PAGE followed by Westernimmunoblotting. Equal fraction volumes from each immunocomplex samplewere analyzed on replicate gels. After electroblotting onto PVDFmembranes, the blots from the replicate gels were probed in parallelwith the monoclonal antibodies 7BD-33-11A, RFAC4, H5C6, and with theIgG_(2a) and IgG₁ isotype controls. In FIG. 7 a the result from thecross-IP experiments in which the material immunoprecipitated by each ofthe test monoclonal antibodies 7BD-33-11A and RFAC4 was analyzed byWestern immunoblotting. In FIG. 7 b the result from the cross-IPexperiments in which the material immunoprecipitated by each of the testmonoclonal antibodies 7BD-33-11A and H5C6 was analyzed by Westernimmunoblotting. Each of the monoclonal antibodies 7BD-33-11A, RFAC4 andH₅C5 cross-reacted with similar antigen(s) immunoprecipitated by7BD-33-11A. In addition, 7BD-33-11A cross reacted, on a Western blot,with similar antigen(s) immunoprecipitated by RFAC4 and H5C6, in therange of 20-80 kDa, but not with the immunocomplexes prepared with theisotype control antibodies. The blots probed with the isotype controlantibodies were completely negative. This data indicated that theepitope recognized by the 7BD-33-11A antibody was contained within theCD63 antigen.

To determine if the cross-reactivity could be due to the same moleculesbeing recognized by all antibodies, or if it was due to the presence ofinteracting molecules with similar mass, immunoprecipitations with theantibody 7BD-33-11A were carried out in conditions of increasing bufferstringency (50 mM Tris pH 7.4, 1% Triton X-100, and varyingconcentrations of NaCl: 0, 150, 500 and 200 mM; and also with RIPAbuffer as described above but containing 500 mM NaCl). The resultingimmunocomplexes were then probed by Western immunoblotting with themonoclonal antibodies 7BD-33-11A, H5C6 and RFAC4 and with the isotypecontrols IgG_(2a) and IgG₁. FIG. 8 showed that varying the stringency ofthe IP conditions did not have any detectable impact on the formation ofthe immunocomplexes, which indicated that the molecule(s) recognized bythe antibody 7BD-33-11A were also recognized by the anti-CD63 antibodiesand vice versa.

To further confirm that 7BD-33-11A was directly binding to the humanCD63 antigen, its reactivity was assessed, by Western immunoblottingagainst lysates of E. coli expressing recombinant fusion polypeptidescontaining the extracellular domains (loops EC1 and EC2) of human CD63.For this work, GST-fusion constructs of the extracellular loops of CD63(loop 1 and loop 2—EC1 and EC2, respectively) were generated bysubcloning the appropriate cDNA fragments into the bacterial expressionvector PGEX-4T-2 (Amersham Biosciences, Piscataway, N.J.). The cDNAfragments encoding the loops were obtained by polymerase chain reactionamplification (PCR), using the full-length human cDNA as a template(clone MGC-8339, American Type Culture Collection Manassas, Va.). ThecDNA encoding the EC1 loop was obtained using the following PCR primers:

5′ primer (EC1_5′), (SEQ ID NO:1) 5′GCCGTGGGATCCGGGGCACAGCTTGTCCTG3′ and3′ primer (EC1_3′), (SEQ ID NO:2) 5′GATGACGAATTCTCACAGAGAGCCAGGGGTAGC3′.The cDNA encoding the EC2 loop was obtained using the following PCRprimers:

5′ primer (EC2_5′), 5′GGCTATGGATCCAGAGATAAGGTGATG3′ (SEQ ID NO:3) and3′ primer (EC2_3′), 5′TACCAGAATTCAATTTTTCCTCAGCCAGCC3′. (SEQ ID NO:4)The conditions for the PCR reactions were as follows: 2 μL of 5′ primer(25 pmol/μL), 2 μL of 3′ primer (25 pmol/μL), 0.2 μL of template DNA(pOTB-CD63, 0.76 mg/mL), and 45.8 μL of PCR SuperMix High Fidelity(Invitrogen, Burlington, ON). The PCR reaction was carried out asfollows: 94° C. for 5 min followed by 30 cycles of: melting at 94° C.for 30 sec, annealing at 55° C. for 30 sec and extension at 72° C. for 1min, per cycle.

After subcloning, the constructs, including a PGEX-4T-2 vector alonenegative control (no cDNA fragment subcloned into the vector), weretransformed into E. coli (strain BL-21). A single ampicillin-resistantcolony from each transformation was grown and the respective insertcDNAs were sequenced. After confirming that the cDNA sequence wascorrect, each of the clones was grown in liquid culture and theexpression of the GST-fusion constructs was induced by addition of 1 mMIPTG (isopropyl-β-D-thiogalactopyranoside) (Gibco-BRL; Rockville, Md.).After a 2 hr incubation, the bacteria culture was centrifuged at 2000 g,for 5 min, at room temperature. The supernatant was discarded and thebacteria pellets were boiled in non-reducing SDS-PAGE sample buffer. Thesamples were then analyzed by SDS-PAGE (5 and 12 percent) polyacrylamidestacking and separating gels respectively) and Western immunoblotting,as previously described. Blot membranes were probed with 7BD-33-11A,H5C6, RFAC4, or with an IgG2a isotype control. The results illustratedby FIG. 9 revealed that 7BD-33-11A specifically recognized loop 2 (aminoacids 108-202) of human CD63 (lane 6 of blot probed with 7BD-33-11A),and does not recognize loop 1 (amino acids 34-52). The specificity ofthe antibody against the bacterial lysate was further confirmed by theobservation that two well-characterized anti-human CD63 antibodies(RFAC4 and H5C6) also recognized a similar size band, only on thelysates from induced E. coli expressing the EC2 fusion polypeptide. Allof the above results demonstrate that 7BD-33-11A recognized and directlybound to human CD63, and specifically to the extracellular regionencompassing amino acids 108-202.

Example 3

As outlined in Ser. No. 10/348,231, the hybridoma cell line 7BD-33-11Awas deposited, in accordance with the Budapest Treaty, with the AmericanType Culture Collection, University Blvd., Manassas, Va. 20110-2209 onJan. 8, 2003, under Accession Number PTA-4890. In accordance with 37 CFR1.808, the depositors assure that all restrictions imposed on theavailability to the public of the deposited materials will beirrevocably removed upon the granting of a patent.

Antibody Production:

7BD-33-11A monoclonal antibody was produced by culturing the hybridomain CL-1000 flasks (BD Biosciences, Oakville, ON) with collections andreseeding occurring twice/week. The antibody was purified according tostandard antibody purification procedures with Protein G Sepharose 4Fast Flow (Amersham Biosciences, Baie d'Urfé, QC).

As previously described in Ser. No. 10/348,231, 7BD-33-11A was comparedto a number of both positive (anti-Fas (EOS9.1, IgM, kappa, 20micrograms/mL, eBioscience, San Diego, Calif.), anti-Her2/neu (IgG1,kappa, 10 microgram/mL, Inter Medico, Markham, ON), anti-EGFR(C225,IgG1, kappa, 5 microgram/mL, Cedarlane, Hornby, ON), Cycloheximide (100micromolar, Sigma, Oakville, ON), NaN₃ (0.1%, Sigma, Oakville, ON)) andnegative (107.3 (anti-TNP, IgG1, kappa, 20 microgram/mL, BD Biosciences,Oakville, ON), G155-178 (anti-TNP, IgG2a, kappa, 20 microgram/mL, BDBiosciences, Oakville, ON), MPC-11 (antigenic specificity unknown,IgG2b, kappa, 20 microgram/mL), J606 (anti-fructosan, IgG3, kappa, 20microgram/mL), IgG Buffer (2%)) controls in a cytotoxicity assay (Table2). Breast cancer (MDA-MB-231 (MB-231), MDA-MB-468 (MB-468), MCF-7),colon cancer (HT-29, SWI116, SW620), lung cancer (NCI H460), ovariancancer (OVCAR), prostate cancer (PC-3), and non-cancer (CCD 27sk, Hs888Lu) cell lines were tested (all from the ATCC, Manassas, Va.). TheLive/Dead cytotoxicity assay was obtained from Molecular Probes (Eugene,Oreg.). The assays were performed according to the manufacturer'sinstructions with the changes outlined below. Cells were plated beforethe assay at the predetermined appropriate density. After 2 days,purified antibody or controls were diluted into media, and then 100microliters were transferred to the cell plates and incubated in a 5percent CO₂ incubator for 5 days. The plate was then emptied byinverting and blotted dry. Room temperature DPBS containing MgCl₂ andCaCl₂ was dispensed into each well from a multi-channel squeeze bottle,tapped three times, emptied by inversion and then blotted dry. 50microliters of the fluorescent calcein dye diluted in DPBS containingMgCl₂ and CaCl₂ was added to each well and incubated at 37° C. in a 5percent CO₂ incubator for 30 minutes. The plates were read in aPerkin-Elmer HTS7000 fluorescence plate reader and the data was analyzedin Microsoft Excel and the results were tabulated in Table 1. The datarepresented an average of four experiments tested in triplicate andpresented qualitatively in the following fashion: 4/4 experimentsgreater than threshold cytotoxicity (+++), 3/4 experiments greater thanthreshold cytotoxicity (++), 2/4 experiments greater than thresholdcytotoxicity (+). Unmarked cells in Table 1 represent inconsistent oreffects less than the threshold cytotoxicity. The 7BD-33-11A antibodydemonstrated cytotoxicity in a breast and prostate tumor cell lineselectively, while having no effect on non-transformed normal cells.7BD-33-11A demonstrated greater killing than the positive controlanti-Fas antibody on the prostate cancer cell line. The chemicalcytotoxic agents induced their expected cytotoxicity while a number ofother antibodies which were included for comparison also performed asexpected given the limitations of biological cell assays. In toto, itwas shown that the 7BD-33-11A antibody has cytotoxic activity against anumber of cancer cell types. The antibody was selective in its activitysince not all cancer cell types were susceptible. Furthermore, theantibodies demonstrated functional specificity since they did notproduce cytotoxicity against non-cancer cell types, which is animportant factor in a therapeutic situation.

TABLE 1 BREAST COLON LUNG OVARY PROSTATE NORMAL MB-231 MB-468 MCF-7HT-29 SW1116 SW620 NCI H460 OVCAR PC-3 CCD 27sk Hs888 Lu 7BD-33-11A −− + − − − − − ++ − − Positive anti-Fas − − +++ − − − − +++ + − +Controls anti-Her2 + − + − − − − + − − − anti-EGFR − +++ + − +++ − − +− + − CHX (100 μM) +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ NaN₃(0.1%) +++ +++ +++ +++ − − +++ +++ +++ − − Negative IgG1 +++ + ControlsIgG2a +++ + IgG2b +++ IgG3 IgG Buffer +

Binding of 7BD-33-11A to the above-mentioned panel of cancer and normalcell lines and to the following additional cancer cell lines; colon(LOVO), pancreatic (BxPC-3), ovarian (ES-2, OCC-1) and prostate (DU-145)and the following additional normal cell line (CCD-12) was assessed byflow cytometry (FACS). Cells were prepared for FACS by initially washingthe cell monolayer with DPBS (without Ca⁺⁺ and Mg⁺⁺). Cell dissociationbuffer (INVITROGEN, Burlington, ON) was then used to dislodge the cellsfrom their cell culture plates at 37° C. After centrifugation andcollection the cells were resuspended in Dulbecco's phosphate bufferedsaline containing MgCl₂, CaCl₂ and 2 or 25 percent fetal bovine serum(FBS) at 4° C. (wash media) and counted, aliquoted to appropriate celldensity, spun down to pellet the cells and resuspended in staining media(DPBS containing MgCl₂ and CaCl₂+/−2 percent FBS) containing 7BD-33-11Aor control antibodies (isotype control or anti-EGFR) at 20 μg/mL on icefor 30 min. Prior to the addition of Alexa Fluor 488-conjugatedsecondary antibody the cells were washed once with wash media. The AlexaFluor 488-conjugated antibody in staining media was then added for 20 to30 min. The cells were then washed for the final time and resuspended instaining media containing 1 μg/mL propidium iodide or 1.5 percentparaformaldehyde. Flow cytometric acquisition of the cells was assessedby running samples on a FACScan using the CellQuest software (BDBiosciences). The forward (FSC) and side scatter (SSC) of the cells wereset by adjusting the voltage and amplitude gains on the FSC and SSCdetectors. The detectors for the three fluorescence channels (FL1, FL2,and FL3) were adjusted by running cells stained with purified isotypecontrol antibody followed by Alexa Fluor 488-conjugated secondaryantibody such that cells had a uniform peak with a median fluorescentintensity of approximately 1-5 units. Live cells were acquired by gatingfor FSC and propidium iodide exclusion (when used). For each sample,approximately 10,000 live cells were acquired for analysis and theresulted presented in Table 2. Table 2 tabulated the mean fluorescenceintensity fold increase above isotype control and is presentedqualitatively as: less than 5 (−); 5 to 50 (+); 50 to 100 (++); above100 (+++) and in parenthesis, the percentage of cells stained.

Representative histograms of 7BD-33-11A antibodies were compiled forFIG. 9. 7BD-33-11A displayed similar binding to cancer lines of breast(MB-231 and MCF-7), colon (HT-29, SW1116 and SW520), lung, ovary,pancreatic and prostate (PC-3) origin and differential binding to one ofthe breast (MB-468), colon (LOVO) and prostate (DU-145) cancer celllines. There was also binding of 7BD-33-11A to non-cancer cells, howeverthat binding did not produce cytotoxicity. This was further evidencethat binding was not necessarily predictive of the outcome of antibodyligation of its cognate antigen, and was a non-obvious finding. Thissuggested that the context of antibody ligation in different cells wasdeterminative of cytoxicity rather than just antibody binding.

TABLE 2 BREAST COLON LUNG OVARY Antibody Isotype MB-231 MB-468 MCF-7HT-29 LOVO SW1116 SW620 NCI H460 ES-2 OCC-1 OVCAR 7BD-33-11A IgG2a, k +− + + − + + + + + + anti-EGFR IgG1, k ++ ++ − + − + − + + + + PANCREATICPROSTATE NORMAL Antibody Isotype BxPC-3 DU-145 PC-3 CCD 27sk CCD-112Hs888 Lu 7BD-33-11A IgG2a, k + − + + + + anti-EGFR IgG1, k + + + + + +

Example 4 Normal Human Tissue Staining

IHC studies were previously conducted to characterize the 7BD-33-11Aantigen distribution in humans (Ser. No. 10/603,006). The currentstudies compared 7BD-33-11A to two antibodies directed against CD63(RFAC4 and H5C6) since the 7BD-33-11A antigen is CD63 as determinedpreviously by biochemical methods. Binding of antibodies to 24 normalhuman tissues was performed using a human normal organ tissue array(Clinomics, Watervliet, N.Y.). All primary antibodies (7BD-33-11A; RFAC4(Cymbus Biotechnology Ltd., Hants, UK) and H5C6 anti-CD63 (BDPharMingen, Oakville, ON); and mouse IgG₁ negative control (Dako,Toronto, ON)) were diluted in antibody dilution buffer (Dako, Toronto,ON) to a concentration of 5 μg/ml (found to be the optimal concentrationin previous optimization steps). The negative control antibody has beenshown to be negative to all mammalian tissues by the manufacturer. Theprocedure for IHC is as follows.

Tissue sections were deparaffinized by drying in an oven at 58° C. for 1hr and dewaxed by immersing in xylene 5 times for 4 min each in Coplinjars. Following treatment through a series of graded ethanol washes(100%-75%) the sections were re-hydrated in water. The slides wereimmersed in 10 mM citrate buffer at pH 6 (Dako, Toronto, Ontario) thenmicrowaved at high, medium, and low power settings for 5 min each andfinally immersed in cold PBS. Slides were then immersed in 3% hydrogenperoxide solution for 6 min, washed with PBS three times for 5 min each,dried, incubated with Universal blocking solution (Dako, Toronto,Ontario) for 5 min at room temperature. 7BD-33-11A, monoclonal mouseanti-CD63 (Cymbus Biotechnology Ltd., Hants, UK or Dako, Toronto,Ontario) or isotype control antibody (directed towards Aspergillus nigerglucose oxidase, an enzyme which is neither present nor inducible inmammalian tissues; Dako, Toronto, Ontario) were diluted in antibodydilution buffer (Dako, Toronto, Ontario) to its working concentration (5μg/mL for each antibody) and incubated overnight for 1 hr at roomtemperature. The slides were washed with PBS 3 times for 5 minutes each.Immunoreactivity of the primary antibodies was detected/visualized withHRP conjugated secondary antibodies as supplied (Dako Envision System,Toronto, Ontario) for 30 minutes at room temperature. Following thisstep the slides were washed with PBS 3 times for 5 minutes each and acolor reaction developed by adding DAB (3,3′-diaminobenzidinetetrahydrachloride, Dako, Toronto, Ontario) chromogen substrate solutionfor immunoperoxidase staining for 10 minutes at room temperature.Washing the slides in tap water terminated the chromogenic reaction.Following counterstaining with Meyer's Hematoxylin (Sigma Diagnostics,Oakville, ON), the slides were dehyrdated with graded ethanols (75-100%)and cleared with xylene. Using mounting media (Dako Faramount, Toronto,Ontario) the slides were coverslipped. Slides were microscopicallyexamined using an Axiovert 200 (Zeiss Canada, Toronto, ON) and digitalimages acquired and stored using Northern Eclipse Imaging Software(Mississauga, ON). Results were read, scored and interpreted by apathologist.

Table 3 presents a summary of the results of 7BD-33-11A and RFAC4 andH5C6 anti-CD63 staining of a test array of normal human tissues. Thestaining of tissues with 7BD-33-11A is similar to that describedpreviously (Ser. No. 10/603,006). It should again be noted that7BD-33-11A showed restricted binding to various cell types but hadbinding to infiltrating macrophages, lymphocytes, and fibroblasts. TheRFAC4 and H5C6 antibodies showed a similar staining pattern incomparison to each other. However, the staining pattern of both RFAC4and H5C6 was quite different than that observed with 7BD-33-11A.Specifically, both RFAC4 and H5C6 antibodies bound to a broader range ofnormal tissues, usually had higher staining intensity in tissues where7BD-33-11A was also positive and bound not only to infiltratingmacrophages, lymphocytes and fibroblasts and but to also to theepithelium in a majority of the tissues (FIG. 11).

Tissues that were positive for 7BD-33-11A were also positive for eitherRFAC4 or H5C6 anti-CD63 antibodies (sometimes with less intensity).Tissues that were negative for 7BD-33-11A were generally not negativefor the RFAC4 or H5C6. These results demonstrated that 7BD-33-11A boundto a smaller subset of the tissues recognized by either the RFAC4 orH5C6 anti-CD63 antibody and within tissues the intensity of staining wasalso sometimes less. These results showed that the antigen for7BD-33-11A was not widely expressed on normal tissues, and that theantibody bound specifically to a limited number of tissues in humans. Italso supported the biochemical evidence that 7BD-33-11A was directedagainst an epitope of CD63, albeit to a different epitope than the onerecognized by either the RFAC4 or H5C6 antibodies used for these IHCstudies.

TABLE 3 Comparison of RFAC4 and H5C6 anti-CD63 and 7BD-33-11A IHC onHuman Normal Tissue Section Tissue 7BD-33-11A RFAC4 H5C6 Aa3 Breast — +(Ductular epithelium and +++ (Ductular epithelium and stromal stromalfibroblasts) fibroblasts) Aa4 Breast +/− (2-3 stromal fibroblasts) +/−(Ductular epithelium and +++ (Stromal fibroblasts) *No staining ofductular epithelium stromal fibroblasts) +/− (Ductular epithelium) Ab3Lung +++ (Macrophages and +++ (Macrophages and +++ (Alveolar epitheliumand fibroblasts at interalveolar septum) fibroblasts at interalveolarseptum) macrophages) Ab4 Lung +++ (Macrophages and +++ (Macrophages and+++ (Macrophages and fibroblasts at fibroblasts at interalveolar septum)fibroblasts at interalveolar septum) interalveolar septum) Ab5 Lung +/−(Macrophages and fibroblasts +++ (Macrophages and +++ (Macrophages andfibroblasts at at interalveolar septum) fibroblasts at interalveolarseptum) interalveolar septum) Ac1 Colon +++ (Lymphocytes and +++(Mucosal epithelium, +++ (Mucosal epithelium, lymphocytes macrophages inlamina propria) *No lymphocytes and macrophages at and macrophages atlamina propria) staining of Mucosal epithelium lamina propria) Ac3 Colon— — +/− (Lymphocytes at lamina propria) Ac4 Colon +++ (Macrophages andfibroblasts +++ (Macrophages and +++ (Mucosal epithelium, lymphocytes atlamina propria) + (Mucosal fibroblasts at lamina propria) + andmacrophages at lamina propria) epithelium) (Mucosal epithelium) Ac5Colon +/− (Macrophages and fibroblasts +++ (Macrophages and +++(Lymphocytes and macrophages in at lamina propria) fibroblasts at laminapropria) + lamina propria) (Mucosal epithelium) Ad1 Prostate +++(Glandular epithelium) +++ (Glandular epithelium) +++ (Glandularepithelium) Ad2 Prostate +++ (Glandular epithelium) +++ (Glandularepithelium) +++ (Glandular epithelium) Ad4 Prostate ++ (Glandularepithelium) +++ (Glandular epithelium) Ad5 Prostate +++ (Glandularepithelium) +++ (Glandular epithelium) +++ (Glandular epithelium) Ae1Kidney — + (Tubular epithelium) ++ (Tubular epithelium) Ae2 Kidney +/−(2-3 interstitial cells) *No ++ (Tubular epithelium) ++ (Tubularepithelium) staining of tubular epithelium Ae3 Kidney +/− (2-3interstitial cells) ++ (Tubular epithelium) ++ (Tubular epithelium) Ae4Liver ++ (Hepatocytes and sinusoidal +++ (Hepatocytes & sinusoidal +++(Hepatocytes, sinusoidal staining staining) staining and bile ductepithelium) and bile ducts) Af1 Liver — ++ (Sinusoidal and bile duct ++(Sinusoidal and bile duct epithelium) epithelium) Af2 Liver — +/−(Hepatocytes and sinusoidal +/− (Hepatocytes and sinusoidal staining)staining) Af3 Lymph node — ++ (Reticular cells) ++ (Reticular cells) Ag1Thyroid — +/− (Follicular cells) +/− (Follicular cells) Ag2 Thyroid +++(Follicular cells) +++ (Follicular cells) +++ (Follicular cells) Ah1Placenta — +++ (Syncytiotrophoblasts & +++ (Syncytiotrophoblasts &basement basement membrane of chorionic membrane of chorionic villi)villi) Ah2 Placenta — +++ (Syncytiotrophoblasts & +++(Syncytiotrophoblasts & basement basement membrane of chorionic membraneof chorionic villi) villi)

Example 5 Human Breast Tumor Tissue Staining

A previous IHC study was undertaken to determine the cancer associationof the 7BD-33-11A antigen with human breast cancers and whether the7BD-33-11A antibody was likely to recognize human cancers (Ser. No.10/603,006). Currently, a comparison was carried out using RFAC4 andH5C6 anti-CD63 and c-erbB-2 anti-Her2 antibodies. A breast cancer tissuearray derived from 50 breast cancer patients and 10 samples derived fromnon-neoplastic breast tissue in breast cancer patients was used (ImgenexCorporation, San Diego, Calif.). The following information was providedfor each patient: age, sex, American Joint Committee on Cancer (AJCC)tumor stage, lymph node, estrogen receptor (ER) and projesteronereceptor (PR) status. The procedure for IHC from Example 4 was followed.All antibodies were used at a working concentration of 5 μg/mL exceptfor the anti-Her2 antibody where a concentration of 1.5 μg/mL was used.

Tables 4, 5 and 6 and 7 provide summaries of 7BD-33-11A, RFAC4 and H5C6anti-CD63 antibody staining of breast cancer tissue arrays. Overall, 36percent of the 50 patients tested were positive for 7BD-33-11A antigencompared to 85 and 94 percent for RFAC4 and H5C6 anti-CD63 antibodiesrespectively. In cases where both 7BD-33-11A and RFAC4 or H5C6 anti-CD63antibodies stained the same tissue, 97 percent of the samples had higherintensity staining with both the RFAC4 and H5C6 anti-CD63 in comparisonto 7BD-33-11A (FIG. 12). For 7BD-33-11A 0 out of 10 and for both RFAC4and H5C6 anti-CD63 antigen 7 out of 8 (2 samples were notrepresentative) normal breast tissue samples from breast cancer patientswere positive, respectively. There was a slight correlation betweenestrogen or progesterone receptor expression and expression of7BD-33-11A antigen; tissues with either receptor expression had slightlyhigher 7BD-33-11A antigen expression. When tumors were analyzed based ontheir stage, or degree to which the cancer advanced, results suggested atrend towards greater positive expression with higher tumor stage for7BD-33-11A. Similar results were obtained with RFAC4. H5C6 also showed avery slight correlation with estrogen or progesterone receptorexpression but there was no apparent correlation with tumor stage.However, for all three antibodies, the results were limited by the smallsample size.

TABLE 4 Human Breast Tumor IHC Summary for 7BD-33-11A Binding Score %positive Total # − +/− + ++ +++ Total positive of total Patient Tumor 5032 10 4 3 1 18 36% Samples Normal 10 10 0 0 0 0 0 0% ER Status ER+ 28 169 1 2 0 12 43% ER− 22 15 3 2 1 1 7 32% Unknown 0 0 0 0 0 0 0 0% PRStatus PR+ 19 9 6 2 2 0 10 53% PR− 30 20 6 2 1 1 10 33% Unknown 1 1 0 00 0 0 0% AJCC Tumor T1 4 4 0 0 0 0 0 0% Stage T2 21 14 3 2 1 1 7 33% T320 11 6 2 1 0 9 45% T4 5 1 3 0 1 0 4 80%

TABLE 5 Human Breast Tumor IHC Summary for RFAC4 Binding Score Total # −+/− + ++ +++ Total positive % positive of total Patient Samples Tumor 477 3 7 16 14 40 85% Normal 8 1 1 0 2 4 7 87.50%   ER Status ER+ 27 1 2 315 6 26 96% ER− 20 6 1 3 4 6 14 70% Unknown 0 0 0 0 0 0 0  0% PR StatusPR+ 18 0 1 2 9 6 18 100%  PR− 28 7 2 4 9 6 21 75% Unknown 1 0 0 0 1 0 1100%  AJCC Tumor Stage T1 4 2 0 1 1 0 2 50% T2 20 4 2 3 6 5 16 80% T3 181 1 2 7 7 17 94% T4 5 0 0 1 2 2 5 100% 

TABLE 6 Human Breast Tumor IHC Summary for H5C6 Binding Score Total # −+/− + ++ +++ Total positive % positive of total Patient Samples Tumor 473 4 8 15 17 44 94% Normal 8 1 1 0 2 4 7 87.50%   ER Status ER+ 27 1 1 68 11 26 96% ER− 20 2 3 2 8 5 18 90% Unknown 0 0 0 0 0 0 0  0% PR StatusPR+ 18 0 0 4 4 10 18 100%  PR− 28 3 4 4 11 6 25 89% Unknown 1 0 0 0 1 01 100%  AJCC Tumor Stage T1 4 0 0 1 2 1 4 100%  T2 20 2 4 3 7 4 18 90%T3 18 1 0 3 4 10 17 94% T4 5 0 0 1 2 2 5 100% 

The 7BD-33-11A staining was specific for cancerous cells in comparisonto normal cells where stromal cells were clearly negative and sheets ofmalignant cells were positive. The cellular localization pattern seenwith the 7BD-33-11A antigen was confined to the cell membrane andcytoplasm. Similar membranous and cytoplasmic staining results wereobtained with the anti-CD63 antibodies, RFAC4 and H5C6 on the breasttumor tissue samples. Additionally, both of these antibodies showed thisstaining localization pattern on normal breast tissue samples whereas7BD-33-11A was negative.

In comparison to c-erbB-2, 7BD-33-11A showed a completely differentstaining profile where 9 out of the 18 breast tumor tissue samples thatwere positive for the 7BD-33-11A antigen were negative for Her2expression indicating a yet unmet targeted therapeutic need for breastcancer patients (Table 8, FIG. 13). There were also differences in theintensity of staining between the breast tumor tissue sections that werepositive for both 7BD-33-11A and Her2; some breast tumor tissue sectionsthat were highly positive for the 7BD-33-11A antigen were only mildlypositive for Her2 and vice versa again illustrating that 7BD-33-11Awould therapeutically target a different cohort of breast cancerpatients. The c-erbB-2 antibody also positively stained one of thenormal breast tissue sections.

These results suggested the antigen for 7BD-33-11A may be expressed byapproximately two thirds of breast cancer patients and half of thosewere completely negative for the Her2 antigen. The staining patternshowed that in patient samples, the antibody is highly specific formalignant cells and the 7BD-33-11A antigen was present on the cellmembrane thereby making it an attractive drugable target. The similaralbeit much more limited staining of 7BD-33-11A versus either the RFAC4or H5C6 anti-CD63 antibody again demonstrates the likelihood of the7BD-33-11A epitope being a more restrictive epitope on CD63.

TABLE 7 Comparison of RFAC4 and H5C6 anti-CD63 and 7BD-33-11A IHC onHuman Tumor and Normal Breast Tissue Data sheet RFAC4 H5C6 7BD-33-11ASec. No. Sex Age Diagnosis Section Score Section Score Section Score 1 F28 Infiltrating duct carcinoma +++ +++ ++ 2 F 71 Solid papillarycarcinoma +++ +++ +/− 3 F 26 Infiltrating duct carcinoma ++ + − 4 F 43Infiltrating duct carcinoma ++ ++ +/− 5 F 39 Infiltrating duct carcinomaNR NR +/− 6 F 46 Ductal carcinoma in situ + + +/− 7 F 47 Infiltratingduct carcinoma +++ +++ + 8 M 67 Infiltrating duct carcinoma +++ +++ + 9F 33 Infiltrating duct carcinoma +++ +++ − 10 F 47 Infiltrating ductcarcinoma ++ ++ − 11 F 49 Invasive lobular carcinoma − − − 12 F 46Infiltrating duct carcinoma ++ ++ − 13 F 39 Infiltrating duct carcinoma++ ++ − 14 F 43 Infiltrating lobular carcinoma +++ +++ +/− 15 F 54Infiltrating lobular carcinoma ++ ++ +/− 16 F 58 Infiltrating ductcarcinoma + ++ +/− 17 F 37 Infiltrating duct carcinoma +++ ++ − 18 F 43Infiltrating duct carcinoma +++ +++ +++ 19 F 51 Infiltrating ductcarcinoma +++ +++ + 20 F 80 Medullary carcinoma ++ ++ − 21 F 36Infiltrating duct carcinoma NR NR − 22 F 59 Infiltrating ductcarcinoma + + − 23 F 34 Ductal carcinoma in situ +++ +++ + 24 F 54Infiltrating duct carcinoma ++ +++ +/− 25 F 47 Infiltrating ductcarcinoma +++ +++ ++ 26 F 53 Infiltrating duct carcinoma ++ ++ − 27 F 59Infiltrating duct carcinoma + + − 28 F 60 Signet ring cell carcinoma F F− 29 F 37 Infiltrating duct carcinoma +++ +++ ++ 30 F 46 Infiltratingduct carcinoma ++ ++ +/− 31 F 35 Infiltrating duct carcinoma − − − 32 F47 Infiltrating duct carcinoma ++ ++ − 33 F 54 Infiltrating ductcarcinoma + + − 34 F 47 Infiltrating duct carcinoma − +/− − 35 F 41Infiltrating duct carcinoma +++ +++ − 36 F 38 Infiltrating ductcarcinoma +++ +++ − 37 F 55 Infiltrating duct carcinoma − +/− − 38 F 65Infiltrating duct carcinoma +/− +/− − 39 M 66 Infiltrating ductcarcinoma − + − 40 F 44 Infiltrating duct carcinoma ++ +++ − 41 F 52Metastatic carcinoma in lymph node ++ ++ − 42 F 32 Metastatic carcinomain lymph node +/− + − 43 F 58 Metastatic carcinoma in lymph node ++ ++++/− 44 F 52 Metastatic carcinoma in lymph node + + − 45 F 58 Metastaticcarcinoma in lymph node − − − 46 F 38 Metastatic carcinoma in lymph node++ +++ − 47 F 45 Metastatic carcinoma in lymph node − ++ − 48 F 45Metastatic carcinoma in lymph node ++ ++ − 49 F 29 Metastatic carcinomain lymph node +/− +/− − 50 F 61 Metastatic carcinoma in lymph node + ++− 51 F 46 Nipple ++ ++ − 52 F 47 Nipple NR NR − 53 F 40 Normal breast+/− +/− − 54 F 43 Normal breast +++ +++ − 55 F 40 Normal breast ++ +++ −56 F 40 Normal breast +++ ++ − 57 F 45 Normal breast NR NR − 58 F 44Normal breast − − − 59 F 37 Normal breast +++ +++ − 60 F 51 Normalbreast +++ +++ − Abbreviations: NR: the sample is not representative andF: the section is folded.

TABLE 8 Comparison of c-erbB-2 anti-Her2 and 7BD-33-11A IHC on HumanTumor and Normal Breast Tissue Data sheet c-erbB-2 7BD-33-11A Sec. No.Sex Age Diagnosis Section Score Section Score 1 F 28 Infiltrating ductcarcinoma + ++ 2 F 71 Solid papillary carcinoma − +/− 3 F 26Infiltrating duct carcinoma +/− − 4 F 43 Infiltrating duct carcinoma +/−+/− 5 F 39 Infiltrating duct carcinoma NR +/− 6 F 46 Ductal carcinoma insitu − +/− 7 F 47 Infiltrating duct carcinoma +++ + 8 M 67 Infiltratingduct carcinoma − + 9 F 33 Infiltrating duct carcinoma +++ − 10 F 47Infiltrating duct carcinoma ++ − 11 F 49 Invasive Lobular carcinoma PD −12 F 46 Infiltrating duct carcinoma − − 13 F 39 Infiltrating ductcarcinoma +++ − 14 F 43 Infiltrating lobular carcinoma − +/− 15 F 54Infiltrating lobular carcinoma − +/− 16 F 58 Infiltrating duct carcinoma− +/− 17 F 37 Infiltrating duct carcinoma +++ − 18 F 43 Infiltratingduct carcinoma − +++ 19 F 51 Infiltrating duct carcinoma + + 20 F 80Medullary carcinoma − − 21 F 36 Infiltrating duct carcinoma NR − 22 F 59Infiltrating duct carcinoma − − 23 F 34 Ductal carcinoma in situ +++ +24 F 54 Infiltrating duct carcinoma + +/− 25 F 47 Infiltrating ductcarcinoma − ++ 26 F 53 Infiltrating duct carcinoma +++ − 27 F 59Infiltrating duct carcinoma + − 28 F 60 Signet ring cell carcinoma − −29 F 37 Infiltrating duct carcinoma +++ ++ 30 F 46 Infiltrating ductcarcinoma − +/− 31 F 35 Infiltrating duct carcinoma − − 32 F 47Infiltrating duct carcinoma +++ − 33 F 54 Infiltrating duct carcinoma −− 34 F 47 Infiltrating duct carcinoma +++ − 35 F 41 Infiltrating ductcarcinoma − − 36 F 38 Infiltrating duct carcinoma ++ − 37 F 55Infiltrating duct carcinoma +/− − 38 F 65 Infiltrating duct carcinoma −− 39 M 66 Infiltrating duct carcinoma − − 40 F 44 Infiltrating ductcarcinoma − − 41 F 52 Metastatic carcinoma in Lymph node − − 42 F 32Metastatic carcinoma in Lymph node − − 43 F 58 Metastatic carcinoma inLymph node ++ +/− 44 F 52 Metastatic carcinoma in Lymph node +++ − 45 F58 Metastatic carcinoma in Lymph node − − 46 F 38 Metastatic carcinomain Lymph node ++ − 47 F 45 Metastatic carcinoma in Lymph node − − 48 F45 Metastatic carcinoma in Lymph node − − 49 F 29 Metastatic carcinomain Lymph node − − 50 F 61 Metastatic carcinoma in Lymph node − − 51 F 46Nipple − − 52 F 47 Nipple +++ − 53 F 40 Normal Breast − − 54 F 43 NormalBreast − − 55 F 40 Normal Breast +/− − 56 F 40 Normal Breast − − 57 F 45Normal Breast − − 58 F 44 Normal Breast − − 59 F 37 Normal Breast − − 60F 51 Normal Breast − −

Example 6 Human Prostate Tissue Staining

To determine whether the 7BD-33-11A antigen was expressed on other humancancer tissues in addition to breast cancer, a multiple human tumortissue array was probed with 7BD-33-11A (Ser. No. 10/603,006; Imgenex,San Diego, Calif.). In furthering those studies, the staining pattern of7BD-33-11A was determined on a human prostate tumor tissue array(Imgenex Corporation, San Diego, Calif.). The staining procedure usedwas the same as the one outlined in Example 4. All antibodies were usedat a working concentration of 5 μg/mL.

As outlined in Table 9, 7BD-33-11A stained 88 percent of human prostatecancers. Although 7BD-33-11A stained the normal tissue sections withhigh intensity as well, there was a higher degree of membranous stainingin the tumor tissue samples in comparison to the normal samples. Therewas one embryonal rhabdomyosarcroma tissue sample that did not stain forthe 7BD-33-11A antigen. There also appeared to be no direct correlationbetween tumor stage and presence of the 7BD-33-11A antigen. However, theresults were limited by the small sample size. Again with 7BD-33-11Athere was both membranous and cytoplasmic staining observed on theprostate tumor tissue samples. However, there was an increase in thedegree of membranous staining relative to that seen with the breasttumor tissue samples (FIG. 14). For the normal prostate tissue samples,this increase in the degree of membranous staining was not observed.

TABLE 9 Human Prostate Tumor IHC Summary for 7BD-33-11A Binding ScoreTotal # − +/− + ++ +++ Total positive % of positive of total Patients'Tumor 51 6 6 6 7 26 45 88% Sample Normal 3 0 0 0 1 2 3 100% TumorAdenocarcinoma 50 5 6 6 7 26 44 88% Subtype embryonal 1 1 0 0 0 0 0 0%Rhabdomyosarcoma Tumor I 1 0 0 0 0 1 1 100% Stage II 11 0 0 1 3 7 11100% III 2 1 0 0 0 1 1 50% IV 32 6 5 5 3 13 26 81%

Therefore, it appeared that the 7BD-33-11A antigen was not solely foundon the membranes of breast cancers but also on the membrane of prostatecancers. These results indicated that 7BD-33-11A has potential as atherapeutic drug in tumor types besides breast.

The preponderance of evidence shows that 7BD-33-11A mediates anti-cancereffects through ligation of a conformational epitope present on avariant of CD63. It has been shown, in Example 2, 7BD-33-11A antibodycan be used to immunoprecipitate the cognate antigen from expressingcells such as MDA-MB-231 cells. Further it could be shown that the7BD-33-11A antibody could be used in detection of cells and/or tissueswhich express a CD63 antigenic moiety which specifically binds thereto,utilizing techniques illustrated by, but not limited to FACS, cell ELISAor IHC.

Thus, it could be shown that the immunoprecipitated 7BD-33-11A antigencan inhibit the binding of 7BD-33-11A to such cells or tissues usingsuch FACS, cell ELISA or IHC assays. Further, as with the 7BD-33-11Aantibody, other anti-CD63 antibodies could be used to immunoprecipitateand isolate other forms of the CD63 antigen, and the antigen can also beused to inhibit the binding of those antibodies to the cells or tissuesthat express the antigen using the same types of assays.

Example 7 In Vivo MDA-MB-231 Preventative Dose Response TumorExperiments

With reference to the data shown in FIGS. 15 and 16, 6 to 8 week old,female SCID mice were implanted with 5 million MDA-MB-231 human breastcancer cells in 100 microliters saline injected subcutaneously in thescruff of the neck. The mice were randomly divided into 4 treatmentgroups of 10. On the day after implantation 0.2, 2.0 or 20 mg/kg of7BD-33-11A or 20 mg/kg IgG isotype control antibody was administeredintraperitoneally at a volume of 300 microliters after dilution from thestock concentration with a diluent that contained 2.7 mM KCl, 1 mMKH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. The antibodies were thenadministered once per week for a period of 7 weeks in the same fashion.Tumor growth was measured about every 7th day with calipers or untilindividual animals reached the CCAC end-points. Body weights of theanimals were recorded for the duration of the study.

At the end of treatment (day 55), the 0.2 mg/kg treatment group hadtumor growth that was 15 percent of the isotype control group. The 85percent reduction in tumor growth in the 0.2 mg/kg 7BD-33-11A treatmentgroup was determined to be a significant difference in comparison to theisotype control as determined by a paired t-test (p<0.0001). Both of the2.0 and 20 mg/kg treatment groups had yet to develop tumors by the endof treatment (day 55). This trend continued on well beyond the treatmentperiod. Treatment with 7BD-33-11A antibody, at all doses, also led to anincrease in survival in comparison to the isotype control treated group.All of the mice in control treated group had died by day 104 (54 daysafter treatment). By contrast, the mice in the 0.2 mg/kg group surviveduntil day 197 (147 days after treatment), 50 percent of the mice in the2.0 mg/kg treatment group were sill alive at day 290 (240 days aftertreatment) and 100 percent of the 20 mg/kg group were also still aliveat also day 290. Therefore, 7BD-33-11A treatment, at all 3 doses,significantly reduced tumor burden and increased survival in comparisonto an isotype control antibody. Treatment at the highest dosedemonstrated the greatest reduction in tumor growth (100 percent) andthe largest increase in survival (all mice are still alive).Consequently, 7BD-33-11A is a potent anti-tumor antibody suggestingpharmacologic and pharmaceutical benefits of this antibody for therapyin other mammals, including man.

Example 8 In Vivo MDA-MB-231 Established Chemotherapy Combination TumorExperiments

With reference to FIGS. 17 and 18, 6 to 8 week old female SCID mice wereimplanted with 5 million MDA-MB-231 human breast cancer cells in 100microlitres saline injected subcutaneously in the scruff of the neck.Tumor growth was measured with calipers every week. When the majority ofthe cohort reached a tumor volume of 100 mm³ (range 48-122 mm³) at 41days post-implantation 8 mice were randomly assigned into each of 4treatment groups. 7BD-33-11A antibody, the chemotherapeutic drugCisplatin, the combination of 7BD-33-11A and Cisplatin or buffer controlwas administered intraperitoneally with 10 or 9 mg/kg of antibody orCisplatin respectively at a volume of 300 microliters after dilutionfrom the stock concentration with a diluent that contained 2.7 mM KCl, 1mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. 7BD-33-11A or buffer controlwas then administered 3 times per week for 10 doses in total in the samefashion until day 64 post-implantation. Cisplatin was administered ondays 1, 3 and 9 of the treatment period. Tumor growth was measured aboutevery seventh day with calipers until day 125 post-implantation or untilindividual animals reached the CCAC end-points. Body weights of theanimals were recorded for the duration of the study. At the end of thestudy all animals were euthanised according to CCAC guidelines.

Using a paired t-test, there was a post-treatment tumor burden reduction(FIG. 16) associated with treatment with either 7BD-33-11A, Cisplatin orthe combination of the two. At day 69 (5 days post-treatment) both7BD-33-11A, Cisplatin and the antibody-drug combination had decreasedmean tumor volumes compared to buffer control treatment; 76 (p<0.001),79 (p<0.001) and 86 percent (p<0.001) respectively. Body weight was usedas a surrogate for well-being. Although both Cisplatin and 7BD-33-11Adisplayed similar tumor suppression, there was not the same degree ofweight loss seen with Cisplatin treatment in comparison to treatmentwith the 7BD-33-11A antibody. There was little difference between thebuffer control and 7BD-33-11A treated groups over the time pointsmonitored. In fact, groups treated with the buffer control and7BD-33-11A showed a slight weight gain after the treatment period. Incontrast, the groups treated with Cisplatin experienced a weight lossthat was especially evident after administration of the final dose. Onday 55 post-implantation (4 days after the final dose of Cisplatin), theCisplatin treated groups showed a 24-30 percent loss in body weight.Therefore both 7BD-33-11A and Cisplatin lowered the tumor burden incomparison to a buffer control in a well-recognized model of humanbreast cancer disease. However, 7BD-33-11A treated animals experiencedbetter well-being than the Cisplatin treatment group as measured by bodyweight. These results suggest pharmacologic, pharmaceutical and qualityof life benefits of this antibody for therapy in other mammals,including man.

Example 9 In Vivo MDA-MB-468 Established Chemotherapy Combination TumorExperiments

With reference to FIGS. 19 and 20, 6 to 8 week old female SCID mice wereimplanted with 2 million MDA-MB-468 human breast cancer cells in 100microlitres saline injected subcutaneously in the scruff of the neck.Tumor growth was measured with calipers every week. When the majority ofthe cohort reached a tumor volume of 100 mm³ (range 11-119 mm³) at 27days post-implantation 8 mice were randomly assigned into each of 4treatment groups. 7BD-33-11A antibody, the chemotherapeutic drugCisplatin, the combination of 7BD-33-11A and Cisplatin or buffer controlwas administered intraperitoneally with 10 or 6 mg/kg of antibody orCisplatin respectively at a volume of 300 microliters after dilutionfrom the stock concentration with a diluent that contained 2.7 mM KCl, 1mM KH₂PO₄, 137 mM NaCl and 20 mM Na₂HPO₄. 7BD-33-11A or buffer controlwas then administered 4 times per week for the first week followed by 3times per week for 11 doses in total in the same fashion until day 50post-implantation. Cisplatin was administered on days 1, 6, 11 and 16 ofthe treatment period. Tumor growth was measured about every seventh daywith calipers until day 66 post-implantation or until individual animalsreached the CCAC end-points. Body weights of the animals were recordedfor the duration of the study. At the end of the study all animals wereeuthanised according to CCAC guidelines.

Using a paired t-test, there was a post-treatment tumor burden reduction(FIG. 18) associated with treatment with either 7BD-33-11A or Cisplatinor the combination of the two. At day 55 (5 days post-treatment) both7BD-33-11A, Cisplatin and the antibody-drug combination had decreasedmean tumor volumes compared to buffer control treatment; 37 (p=0.3958),95 (p=0.024) and 97 percent (p=0.017) respectively. Body weight was usedas a surrogate for well-being. Although both 7BD-33-11A and, to agreater extent, Cisplatin displayed tumor suppression, there was not thesame degree of weight loss seen with 7BD-33-11A antibody treatment incomparison to Cisplatin treatment. There was little difference betweenthe buffer control and the 7BD-33-11A treated groups over the timepoints monitored. In fact, groups treated with the buffer control and7BD-33-11A showed some slight weight gain during the treatment period.In contrast, the groups treated with Cisplatin experienced a weight lossthat was especially evident after the final dose of Cisplatin wasadministered. On day 48 post-implantation (4 days after the final doseof Cisplatin), the Cisplatin treated groups showed a 20 percent loss inbody weight. Therefore both 7BD-33-11A and Cisplatin lowered the tumorburden in comparison to a buffer control in another well-recognizedmodel of human breast cancer disease. However, 7BD-33-11A treatedanimals experienced better well-being than the Cisplatin treatment groupas measured by body weight. In all, these results in which 7BD-33-11Aproduced significant benefits (improved survival, decreased tumor burdenin comparison to control treatment, and better tolerability incomparison to chemotherapy) in multiple models of human cancer suggestpharmacologic, pharmaceutical and quality of life benefits of thisantibody for therapy in other mammals, including man.

The preponderance of evidence shows that 7BD-33-11A mediates anti-cancereffects through ligation of an epitope present on extracellular loop 2on CD63. It has been shown, in Example 2, 7BD-33-11A antibody can beused to immunoprecipitate the cognate antigen from expressing cells suchas MDA-MB-231 cells. Further it could be shown that the 7BD-33-11Aantibody could be used in detection of cells and/or tissues whichexpress a CD63 antigenic moiety which specifically binds thereto,utilizing techniques illustrated by, but not limited to FACS, cell ELISAor IHC.

Thus, it could be shown that the immunoprecipitated 7BD-33-11A antigencan inhibit the binding of 7BD-33-11A to such cells or tissues usingFACS, cell ELISA or IHC assays. Further, as with the 7BD-33-11Aantibody, other anti-CD63 antibodies could be used to immunoprecipitateand isolate other forms of the CD63 antigen, and the antigen can also beused to inhibit the binding of those antibodies to the cells or tissuesthat express the antigen using the same types of assays.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementof parts herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Anyoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for treating a patient suffering from a cancerous diseasecomprising: administering to said patient an anti-cancer antibody orfragment thereof produced in accordance with a method for the productionof anti-cancer antibodies which are useful in treating a cancerousdisease, said antibody or fragment thereof characterized as beingcytotoxic against cells of a cancerous tissue, and being essentiallybenign to non-cancerous cells; wherein said antibody or fragment thereofis placed in admixture with a pharmaceutically acceptable adjuvant andis administered in an amount effective to mediate treatment of saidcancerous disease; said antibody being an isolated monoclonal antibodyor antigen binding fragment thereof which binds to an antigenic moietyexpressed by said cancerous tissue, said antigenic moiety characterizedas being bound by an antibody having identifying characteristics of amonoclonal antibody encoded by a clone deposited with the ATCC asPTA-4890.
 2. The method for treating a patient suffering from acancerous disease in accordance with claim 1, wherein said antibody orfragment thereof is humanized or chimerized.
 3. The method for treatinga patient suffering from a cancerous disease in accordance with claim 1comprising: conjugating said antibody or antigen binding fragmentthereof with a member selected from the group consisting of toxins,enzymes, radioactive compounds, and hematogenous cells, thereby formingan antibody conjugate; and administering said antibody conjugate orconjugated fragments thereof to said patient; wherein said antibodyconjugate or conjugated fragments are placed in admixture with apharmaceutically acceptable adjuvant and are administered in an amounteffective to mediate treatment of said cancerous disease.
 4. The methodof claim 3, wherein said antibody or fragment thereof is humanized orchimerized.
 5. The method for treating a patient suffering from acancerous disease in accordance with claim 1 wherein: the cytotoxicityof said antibody or fragment thereof is mediated through antibodydependent cellular toxicity.
 6. The method for treating a patientsuffering from a cancerous disease in accordance with claim 1 wherein:the cytotoxicity of said antibody or fragment thereof is mediatedthrough complement dependent cellular toxicity.
 7. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 1 wherein: the cytotoxicity of said antibody or fragment thereofis mediated through catalyzing of the hydrolysis of cellular chemicalbonds.
 8. The method for treating a patient suffering from a cancerousdisease in accordance with claim 1 wherein: the cytotoxicity of saidantibody or fragment thereof is mediated through producing an immuneresponse against putative cancer antigens residing on tumor cells. 9.The method for treating a patient suffering from a cancerous disease inaccordance with claim 1 wherein: the cytotoxicity of said antibody orfragment thereof is mediated through targeting of cell membrane proteinsto interfere with their function.
 10. The method for treating a patientsuffering from a cancerous disease in accordance with claim 1 wherein:the cytotoxicity of said antibody or fragment thereof is mediatedthrough production of a conformational change in a cellular proteineffective to produce a signal to initiate cell-killing.
 11. The methodfor treating a patient suffering from a cancerous disease in accordancewith claim 1 wherein: said method of production utilizes a tissue samplecontaining cancerous and non-cancerous cells obtained from a particularindividual.
 12. A method for treating a patient suffering from acancerous disease comprising: administering to said patient an antibodyor antigen binding fragment thereof produced in accordance with a methodfor the production of anti-cancer antibodies which are useful intreating a cancerous disease, said antibody being cytotoxic againstcells of a cancerous tissue, and essentially benign to non-cancerouscells; wherein said antibody is the isolated monoclonal antibody encodedby the clone deposited with the ATCC as PTA-4890 or an antigen bindingfragment thereof, and is placed in admixture with a pharmaceuticallyacceptable adjuvant and is administered in an amount effective tomediate treatment of said cancerous disease.
 13. The method for treatinga patient suffering from a cancerous disease in accordance with claim12, wherein said antibody or fragment thereof is humanized orchimerized.
 14. The method for treating a patient suffering from acancerous disease in accordance with claim 12 comprising: conjugatingsaid antibody or fragment thereof with a member selected from the groupconsisting of toxins, enzymes, radioactive compounds, and hematogenouscells, whereby an antibody conjugate is formed; and administering saidantibody conjugates or fragments thereof to said patient; wherein saidconjugated antibodies are placed in admixture with a pharmaceuticallyacceptable adjuvant and are administered in an amount effective tomediate treatment of said cancerous disease.
 15. The method of claim 14,wherein said antibody or fragment thereof is selected from said subsetare humanized or chimerized.
 16. The method for treating a patientsuffering from a cancerous disease in accordance with claim 12 wherein:the cytotoxicity of said antibody or fragment thereof is mediatedthrough antibody dependent cellular toxicity.
 17. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 12 wherein: the cytotoxicity of said antibody or fragment thereofis mediated through complement dependent cellular toxicity.
 18. Themethod for treating a patient suffering from a cancerous disease inaccordance with claim 12 wherein: the cytotoxicity of said antibody orfragment thereof is mediated through catalyzing of the hydrolysis ofcellular chemical bonds.
 19. The method for treating a patient sufferingfrom a cancerous disease in accordance with claim 12 wherein: thecytotoxicity of said antibody or fragment thereof is mediated throughproducing an immune response against putative cancer antigens residingon tumor cells.
 20. The method for treating a patient suffering from acancerous disease in accordance with claim 12 wherein: the cytotoxicityof said antibody or fragment thereof is mediated through targeting ofcell membrane proteins to interfere with their function.
 21. The methodfor treating a patient suffering from a cancerous disease in accordancewith claim 12 wherein: the cytotoxicity of said antibody or fragmentthereof is mediated through production of a conformational change in acellular protein effective to produce a signal to initiate cell-killing.22. The method for treating a patient suffering from a cancerous diseasein accordance with claim 12 wherein: said method of production utilizesa tissue sample containing cancerous and non-cancerous cells obtainedfrom a particular individual.
 23. A process for mediating cytotoxicityof a human tumor cell which expresses a CD63 antigenic moiety on thecell surface comprising: contacting said tumor cell with an isolatedmonoclonal antibody or antigen binding fragment thereof, said antibodyor antigen binding fragment thereof being an isolated monoclonalantibody or antigen binding fragment thereof which binds to saidexpressed CD63 antigenic moiety, said antigenic moiety characterized asbeing bound by an antibody having the identifying characteristics of amonoclonal antibody encoded by the clone deposited with the ATCC asPTA-4890, whereby cell cytotoxicity occurs as a result of said binding.24. The process of claim 23 wherein said isolated antibody or antigenbinding fragments thereof are humanized or chimerized.
 25. The processof claim 23 wherein said isolated antibody or antigen binding fragmentsthereof are conjugated with a member selected from the group consistingof cytotoxic moieties, enzymes, radioactive compounds, and hematogenouscells, whereby an antibody conjugate is formed.
 26. The process of claim23 wherein said isolated antibody or antigen binding fragments thereofare humanized or chimerized.
 27. The process of claim 23 wherein saidisolated antibody or antigen binding fragments thereof are murine. 28.The process of claim 23 wherein the human tumor tissue sample isobtained from a tumor originating in a tissue selected from the groupconsisting of colon, ovarian, lung, prostate and breast tissue.
 29. Abinding assay to determine a presence of cells which express a CD63antigenic moiety which specifically binds to an isolated monoclonalantibody encoded by the clone deposited with the ATCC as PTA-4890 or anantigen binding fragment thereof comprising: providing a cell sample;providing an isolated monoclonal antibody or antigen binding fragmentthereof, said antibody or antigen binding fragment thereof being anisolated monoclonal antibody or antigen binding fragment thereof whichbinds to said expressed CD63 antigenic moiety, said antigenic moietycharacterized as being bound by an antibody having the identifyingcharacteristics of a monoclonal antibody encoded by the clone depositedwith the ATCC as PTA-4890; contacting said isolated monoclonal antibodyor antigen binding fragment thereof with said cell sample; anddetermining binding of said isolated monoclonal antibody or antigenbinding fragment thereof with said cell sample; whereby the presence ofcells which express a CD63 antigenic moiety which specifically binds tosaid isolated monoclonal antibody or antigen binding fragment thereof isdetermined.
 30. The binding assay of claim 29 wherein the cell sample isobtained from a tumor originating in a tissue selected from the groupconsisting of colon, ovarian, lung, prostate and breast tissue.
 31. Aprocess of isolating or screening for cells in a sample which express aCD63 antigenic moiety which specifically binds to an isolated monoclonalantibody or antigen binding fragment thereof, said antigenic moietycharacterized as being bound by an antibody having the identifyingcharacteristics of a monoclonal antibody encoded by the clone depositedwith the ATCC as PTA-4890 comprising: providing a cell sample; providingan isolated monoclonal antibody or antigen binding fragment thereof,said antibody or antigen binding fragment thereof being an isolatedmonoclonal antibody or antigen binding fragment thereof which binds tosaid expressed CD63 antigenic moiety, said antigenic moietycharacterized as being bound by an antibody having the identifyingcharacteristics of a monoclonal antibody encoded by the clone depositedwith the ATCC as PTA-4890; contacting said isolated monoclonal antibodyor antigen binding fragment thereof with said cell sample; anddetermining binding of said isolated monoclonal antibody or antigenbinding fragment thereof with said cell sample; whereby said cells whichexpress a CD63 antigenic moiety which specifically binds to an isolatedmonoclonal antibody encoded by the clone deposited with the ATCC asPTA-4890, or antigen binding fragment thereof are isolated by saidbinding and their presence in said cell sample is confirmed.
 32. Theprocess of claim 31 wherein the cell sample is obtained from a tumororiginating in a tissue selected from the group consisting of colon,ovarian, lung, prostate and breast tissue.
 33. A method of extendingsurvival and/or delaying disease progression by treating a human tumorin a mammal, wherein said tumor expresses an antigen which specificallybinds to a monoclonal antibody or antigen binding fragment thereof whichhas the identifying characteristics of a monoclonal antibody encoded bya clone deposited with the ATCC as accession number PTA-4890 comprisingadministering to said mammal said monoclonal antibody in an amounteffective to reduce said mammal's tumor burden, whereby diseaseprogression is delayed and/or survival is extended.
 34. The method ofclaim 33 wherein said antibody is conjugated to a cytotoxic moiety. 35.The method of claim 33 wherein said cytotoxic moiety is a radioactiveisotope.
 36. The method of claim 33 wherein said antibody activatescomplement.
 37. The method of claim 33 wherein said antibody mediatesantibody dependent cellular cytotoxicity.
 38. The method of claim 33wherein said antibody is a murine antibody.
 39. The method of claim 33wherein said antibody is a humanized antibody
 40. The method of claim 33wherein said antibody is a chimerized antibody.